Unifying mechanism and methods to prevent cancer and neurodegenerative diseases

ABSTRACT

The present invention relates to methods for preventing the development of cancer or neurodegenerative diseases by administering N-Acetylcysteine (NAC), melatonin, or a combination thereof. The present invention also relates to methods for diagnosing cancer and/or neurdegenerative disease by detecting or determining the amount of dopamine metabolites, 4-CE, 2-CE, methylation of CE or CE-Q conjugates.

RELATED APPLICATION

This application is a continuation-in-part under 35 U.S.C. 111(a) ofInternational Application No. PCT/US03/007686 filed Mar. 12, 2003 andpublished in English as WO 03/077900 on Sep. 25, 2003, which claimedpriority from Provisional Application No.60/364,544, filed Mar. 14,2002, which applications and publication are incorporated herein byreference.

GOVERNMENT FUNDING

The invention described herein was made with government support underGrant Number PO1CA49210 and RO1CA49917 awarded by the National CancerInstitute, NIH. The United States Government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

Cancer is a disease that begins with mutation of critical genes:oncogenes and tumor suppressor genes. Mutation of critical genes allowsfor a cancer cell to evolve and ultimately results in pathogenicreplication (a loss of normal regulatory control leading to excessivecell proliferation) of various given types of cells found in the humanbody. Conventional cancer treatments have focused mainly on killingcancerous cells. Such treatments threaten noncancerous cells, inherentlyare stressful to the human body, produce many side effects, and are ofuncertain efficacy. More important, such treatment regimens are notnecessarily directed toward the actual root of the cancer problem or itsprevention.

Other diseases are associated with excessive cell death. For example,diseases associated with the loss of neurons in different regions of thecentral nervous system (CNS), including, for example, brain tissue andthe spinal cord, such as Alzheimer's disease, amyotrophic lateralsclerosis (“ALS” or “Lou Gehrig's disease”), Parkinson's disease,Huntington's disease, brain aging, Friedreich's ataxia, multiplesclerosis, diabetic necrosis, ischaemia, and stroke. These types ofdiseases are exemplary of diseases and disorders collectively referredto as “neurodegenerative diseases.” Treatment and prevention ofneurodegenerative disorders remains elusive in that many proposedtreatment methods are not practical since exogenous administration ofnumerous putative therapeutics is not efficacious due to their generalinability to cross the blood-brain barrier.

Thus, there is a need in the art for therapeutic methods to prevent orreduce the risk of the development of cancer and/or the development ofneurodegenerative diseases.

SUMMARY OF THE INVENTION

Applicant has discovered that oxidation of the carcinogenic 4-hydroxycatechol estrogens (CE) of estrone (E₁) and estradiol (E₂) to catecholestrogen-3,4-quinones (CE-3,4-Q) results in electrophilic intermediatesthat covalently bind to DNA to form depurinating adducts at the N-7 ofguanine and N-3 of adenine by 1,4-Michael addition. The resultantapurinic sites in critical genes can generate mutations that mayinitiate various human cancers. As such, the endogenous quinones,including CE-3,4-Q, may be endogenous tumor initiators. As yet, thereare no treatment methods available that are specifically directed towardpreventing the association of the metabolic intermediates, such asendogenous (that which has originated or been produced within anorganism, tissue, or cell) quinones, with DNA, and thus, preventingexcessive cell growth and the development/formation of cancer.

Applicant has also discovered that the catecholamine dopamine and themetabolite catechol (1,2-dihydroxybenzene) of the leukemogenic benzenecan be oxidized to their quinones which react with DNA to formpredominantly analogous depurinating adducts. In the case ofdepurinating adducts resulting from oxidization of dopamine to itsquinone, the resultant apurinic sites in critical genes can generatemutations that may initiate brain cancer and/or neurodegenerativediseases.

Therefore, Applicant has discovered that apurinic sites formed bydepurinating adducts are converted into tumor-initiating orneurodegenerative-initiating mutations by error-prone repair. Thus,Applicant has discovered a unifying molecular mechanism of initiationfor many cancers and neurodegenerative diseases. Using this unifyingmolecular mechanism, Applicant has also designed strategies to assessrisk and to prevent such diseases.

Applicant has also discovered that N-acetylcysteine (NAC) is capablepreventing the formation of depurinating adducts (endogenous tumorinitiators and/or neurodegenerative initiators) by aiding in theremoval, detoxification and/or sequestration of, for example, catecholquinones and/or the oxidation products of benzene and dopamine prior totheir association with DNA. Applicant has further discovered that NACmay be useful to prevent the formation of quinones.

Applicant has further discovered that melatonin is useful in theprevention of the formation of depurinating adducts, particularly thoseformed in the brain due to dopamine oxidation and the resultantproduction of its quinone.

Thus, Applicant has discovered that the use of NAC and/or melatonin canprevent or diminish the formation of depurinating adducts, andtherefore, prevent and/or treat cancer and/or neurodegenerativedisorders. Further, it is believed that practice of the invention will,at least in part, influence and control cellular mortality by allowingthe cell to maintain a lower level of endogenous quinones (that have theability to bind DNA and form depurinating adducts) and thus, allow thecell to follow a normal apoptotic pathway (programmed cell death, suchas that signaled by the nuclei in normally functioning human and animalcells when age or state of cell health and condition dictates).

Accordingly, the present invention provides pharmaceutical compositionsand methods to treat and/or prevent cancer and neurodegenerativediseases and for reducing cancer and neurodegenerative diseasemortality. The present invention is further directed to methods ofutilizing N-acetylcysteine (NAC) and melatonin to treat, prevent, and/orreduce the risk of cancer and neurodegenerative diseases and disorders,to reduce the formation of DNA adducts by reactive electrophilicestrogen metabolites, and/or to reduce the formation of DNA adducts byreactive electrophilic dopamine metabolites.

The present invention also relates to a therapeutic method ofpreventing, treating or reducing the risk of a pathological condition orsymptom in a mammal, including a human, which is suffering or may sufferfrom said condition, wherein production of quinones is implicated andantagonism of such production or removal of such quinones is desired,comprised of administering to a mammal an effective amount of NAC,melatonin, a physiologically acceptable salt thereof, or a combinationthereof. Therefore, the present invention provides a method formodulating quinone production (e.g., CE-3,4-Q or the quinone ofdopamine) or altering the amount of quinones present in a mammal. Alsoprovided is a therapeutic method for preventing or treating apathological condition or symptom in a mammal, such as a human, whereinthe production of depurinating DNA adducts from the action of endogenousquinones is implicated and antagonism of such action is desired,comprised of administering to a mammal in need of such therapy, aneffective amount of NAC, melatonin, a pharmaceutically acceptable saltthereof, or a combination thereof.

Further provided is a method of treating or preventing a neoplastic orneurodegenerative condition or both conditions in a subject comprisingadministering an effective amount of NAC, melatonin, a physiologicallyacceptable salt thereof, or a combination thereof. The combinationtreatment method provides for simultaneous, sequential or separate usein treating such conditions.

The present invention also relates to a method for identifying an agentuseful to prevent, reduce the risk, or treat cancer comprised ofcontacting a host cell with E₂ and a candidate agent and determiningwhether the candidate agent reduces the amount of CE-3,4-Q ordepurinating adducts in the cell compared to a control. Another methodfor identifying an agent useful to prevent or reduce the risk of canceror neurodegenerative disease comprises incubating DNA with a catecholestrogen quinone or a catecholamine dopamine quinone and a candidateagent and determining whether the candidate agent reduces theassociation of the quinone with DNA. The invention also provides agentsidentified by such methods.

Also provided is a method for determining the risk of developing canceror a neurodegenerative disease in a mammal comprising determining theamount of endogenous quinone present in a biological test sample, suchas blood, urine or other body fluid (including spinal fluid) or a tissuebiopsy, and comparing the determined amount to an amount present in anormal sample, wherein an increase in amount of quinone correlates withthe risk of developing cancer (e.g., breast cancer), and/or aneurodegenerative disease.

The invention further provides a method for detecting cancer and/orneurodegenerative disease in a mammal, preferably a human. The methodcomprises subjecting a physiological sample from a human to analyticaldetection to determine the presence or amount of dopamine metabolites(such as dopamine quinones), 4-CE, 2-CE, methylation of CE and/or CE-Qconjugates. The presence or amount of dopamine metabolites, 4-CE, 2-CE,methylation of CE and/or CE-Q conjugates is then compared to an amountpresent in a control sample, wherein an increase in the amount ofdopamine metabolites, 4-CE, methylation of CE and/or CE-Q conjugatescorrelates to the presence or absence of cancer and/or neurodegenerativedisease. Also provided is a diagnostic method for detecting dopaminemetabolites, 4-CE, 2-CE, methylation of CE and/or CE-Q conjugates.

The presence or amount of dopamine metabolites, 4-CE, 2-CE, methylationof CE and/or CE-Q conjugates that changes over time can indicate theprogression or remission of cancer, such as breast cancer, orneurodegenerative disease, as well as the presence of previouslyundiagnosed metastatic or neurodegenerative disease. Thus, the presentinvention provides a method for monitoring the course, progression orremission of cancer, such as breast cancer, and neurodegenerativedisease. This method comprises analyzing a physiological sample byanalytical methods. The presence or amount of dopamine metabolites,4-CE, 2-CE, methylation of CE and/or CE-Q conjugates is detected ordetermined. At least one point later in time, another sample is takenand the amount of dopamine metabolites, 4-CE, 2-CE, methylation of CEand/or CE-Q conjugates is determined. The amounts of dopaminemetabolites, 4-CE, 2-CE, methylation of CE and/or CE-Q conjugates,obtained at least at two different time points, are compared.

The methods of the invention also optionally comprise administering anagent that induces the protective enzyme quinone reductase.

The methods of the invention also optionally comprise administering anagent that inhibits CYP1B1.

The methods of the invention also optionally comprise administeringlipoic acid or a pharmaceutically acceptable salt thereof.

The methods of the invention also optionally comprise administeringresveratrol or a pharmaceutically acceptable salt thereof.

The methods of the invention also optionally comprise administeringlipoic acid and resveratrol or pharmaceutically acceptable saltsthereof.

The present invention also provides pharmaceutical compositions whichcomprise an effective amount of NAC and an effective amount of melatoninor a physiologically acceptable salt thereof, together with one or morephysiologically acceptable carriers or excipients. Such a composition isuseful, for example, to treat and/or prevent cancer and/orneurodegenerative diseases, as well as other diseases that are effectedby the activity of quinones (e.g., formation of genetic lesions). Theinvention also provides a pharmaceutical composition comprising 1) NACor a physiologically acceptable salt thereof, 2) melatonin or aphysiologically acceptable salt thereof, 3) optionally one or moreagents that induce quinone reductase, and 4) one or more physiologicallyacceptable carriers or excipients. The compositions of the invention canalso optionally comprise an agent that inhibits CYP1B1.

The invention also provides the use of NAC and/or melatonin to prepare amedicament useful to treat cancer and/or neurodegenerative diseases.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the formation of stable and depurinating DNA adducts, andgeneration of apurinic sites.

FIG. 2 depicts the formation, metabolism, conjugation and DNA adducts ofestrogens.

FIG. 3 demonstrates redox cycling of catechol estrogen semiquinones andquinones: DNA damage and formation of lipid hydroperoxides.

FIGS. 4A-D depict H-ras mutations induced by DB[a,l]P or its metabolite,anti-DB[a,l]PDE. Wild type sequences and nucleotide numbers (GenBankaccession No. U89950) are indicated below and mutations are indicatedabove the line. (A) PCR artifact mutations induced in untreated skin DNAand in a cloned H-ras gene (pWT) treated with anti-DB[a,l]PDE or withacid. Under the treatment conditions, anti-DB[a,l]PDE induces 1 adductper 1000 bases and acid induces 1 depurination per 170 bases(Chakravarti, D. Et al., Mutat. Res., 456, 17-32 (2000)). (B) H-rasmutations in mouse skin DNA after treatment with 200 nmol DB[a,l]P in100 μL acetone. At 12 h-1 d, the spectra contained mostly A/T to G/Cmutations. At days 2 and 3, multiple codon 61 mutations were observed.At 4 d, no clear pattern of mutations could be determined. At days 5 and6, multiple codon 52 (CTA to CCA) mutations were observed. Few mutationswere observed at day 9. ∇, insertion. (C) H-ras mutations in mouse skinDNA after treatment with 200 nmol of anti-DB[a,l]PDE in 100 μL acetone.Fifty to sixty percent of mutations between days 1 to 4 were A/T to G/Cmutations. (D) H-ras mutations after TDG treatment of DNA fromanti-DB[a,l]PDE-treated pWT and from DB[a,l]P- oranti-DB[a,l]PDE-treated mouse skin. TDG treatment resulted in drasticreduction of A/T to G/C mutations and the observation of multiple codon61 (CAA to CTA) mutations at day 1. These mutations were also observedat days 2 and 3. In addition, at days 2 and 3, multiple codon 13 (GGC toGTC) mutations were observed.

FIG. 5 depicts a proposed pathway of formation of A to G mutations byerror-prone base excision repair of carcinogen-induced apurinic sitesand the detection of the resulting G.T heteroduplexes by the TDG-PCRtechnique. The conversion of G.T heteroduplexes into G. apyrimidinicsites results in a drastic reduction in the formation of A/T to G/Cmutations. G.T heteroduplexes are converted into fixed mutations (G.Cand A.T pairs) by one round of replication.

FIG. 6 demonstrates sequence similarity among sites of DB[a,l]P-inducedmutations in H-ras DNA of mouse skin at day 1. A putative conservedsequence is shaded. The mutated base is underlined. The italicizedsequence (A³¹⁴→G mutation) is from the bottom strand.

FIGS. 7A-C depict H-ras mutations induced by E₂-3,4-Q. (A) PCR artifactmutations induced in untreated skin DNA and in a cloned H-ras gene (pWT)treated with E₂-2,3-Q or with E₂-3,4-Q. (B) H-ras mutations in mouseskin DNA after treatment with 200 nmol E₂-3,4-Q in 100 mL ofacetone/ethanol (70:30). The spectra contained mostly A/T to G/Cmutations. (C) H-ras mutations after TDG treatment of DNA fromE₂-3,4-Q-treated mouse skin. TDG treatment resulted in drastic reductionof A/T to G/C mutations in 6 h and 12 h samples, but not in 1 d and 3 dsamples. This suggests that these mutations were in the form of G.Theteroduplexes between 6-12 h, but were converted into fixed mutationsafter that.

FIGS. 8A-B demonstrate a unifying mechanism of activation and formationof DNA adducts. (A) Natural and synthetic estrogens, and (B) Benzene anddopamine.

FIG. 9 demonstrates metabolism of 4-OHE₁(E₂) and formation ofdepurinating DNA adducts.

FIG. 10 depicts the synthesis of CAT-4-N7Gua and CAT-4-N3Ade by reactionof CAT quinone with dG or Ade.

FIG. 11 demonstrates the synthesis of DA (NADA)-6-N7Gua and DA(NADA)-6-N3Ade by reaction of DA (NADA) quinone with dG or Ade.

FIG. 12 depicts the metabolism of DA to form neuromelanin ordepurinating DNA adducts.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based upon the discovery of a unifyingmechanism, namely, formation of catechol quinones and reaction with DNAby 1,4-Michael addition to yield depurinating adducts that could giverise to cancers and/or neurodegenerative diseases. The present inventiontherefore provides pharmaceutical compositions and methods to preventand/or treat cancer and/or neurodegenerative diseases resulting from theformation of quinones and/or the reaction of such quinones with DNA by1,4-Michael addition yielding depurinating adducts.

Compositions of NAC and/or Melatonin for Therapeutic Use

Therapeutic and/or effective amounts of NAC, melatonin, or a combinationthereof are amounts which are effective to: prevent the development,further development, or reduce the risk of development of cancer and/orneurodegenerative diseases; reduce the formation of DNA adducts byendogenous reactive electrophilic estrogen metabolites; and/or reducethe formation of DNA adducts by endogenous reactive electrophilicdopamine metabolites. Such effects are achieved while exhibiting littleor no adverse effects on normal, healthy tissues or cells or whileexerting negligible or manageable adverse side effects on normal,healthy tissues or cells of the mammal.

Administration of NAC and/or melatonin as salts may be appropriate.Examples of pharmaceutically acceptable salts are organic acid additionsalts formed with acids which form a physiological acceptable anion, forexample, tosylate, methanesulfonate, acetate, citrate, malonate,tartarate, succinate, benzoate, ascorbate, α-ketoglutarate, andα-glycerophosphate. Suitable inorganic salts may also be formed,including hydrochloride, sulfate, nitrate, bicarbonate, and carbonatesalts.

Pharmaceutically acceptable salts may be obtained using standardprocedures well known in the art, for example by reacting a sufficientlybasic compound such as an amine with a suitable acid affording aphysiologically acceptable anion. Alkali metal (for example, sodium,potassium or lithium) or alkaline earth metal (for example calcium)salts of carboxylic acids can also be made.

NAC and/or melatonin can be formulated as pharmaceutical compositionsand administered to a mammalian host, such as a human patient in avariety of forms adapted to the chosen route of administration, i.e.,orally or parenterally, by intravenous, intramuscular, topical orsubcutaneous routes.

Thus, NAC and/or melatonin may be systemically administered, e.g.,orally, in combination with a pharmaceutically acceptable vehicle suchas an inert diluent or an assimilable edible carrier. They may beenclosed in hard or soft shell gelatin capsules, may be compressed intotablets, or may be incorporated directly with the food of the patient'sdiet. For oral therapeutic administration, the active compound may becombined with one or more excipients and used in the form of ingestibletablets, buccal tablets, troches, capsules, elixirs, suspensions,syrups, wafers, and the like. Such compositions and preparations shouldcontain at least 0.1% of active compound. The percentage of thecompositions and preparations may, of course, be varied and mayconveniently be between about 2 to about 60% of the weight of a givenunit dosage form. The amount of active compound in such therapeuticallyuseful compositions is such that an effective dosage level will beobtained.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like. A syrup or elixir maycontain the active compound, sucrose or fructose as a sweetening agent,methyl and propylparabens as preservatives, a dye and flavoring such ascherry or orange flavor. Of course, any material used in preparing anyunit dosage form should be pharmaceutically acceptable and substantiallynon-toxic in the amounts employed. In addition, the active compound maybe incorporated into sustained-release preparations and devices.

NAC and/or melatonin may also be administered intravenously orintraperitoneally by infusion or injection. Solutions of the activecompound or its salts can be prepared in water, optionally mixed with anontoxic surfactant. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, triacetin, and mixtures thereof and inoils. Under ordinary conditions of storage and use, these preparationscontain a preservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. In all cases, theultimate dosage form should be sterile, fluid and stable under theconditions of manufacture and storage. The liquid carrier or vehicle canbe a solvent or liquid dispersion medium comprising, for example, water,ethanol, a polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycols, and the like), vegetable oils, nontoxic glycerylesters, and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the formation of liposomes, by themaintenance of the required particle size in the case of dispersions orby the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfilter sterilization. In the case of sterile powders for the preparationof sterile injectable solutions, the preferred methods of preparationare vacuum drying and the freeze drying techniques, which yield a powderof the active ingredient plus any additional desired ingredient presentin the previously sterile-filtered solutions.

For topical administration, NAC and/or melatonin may be applied in pureform, i.e., when they are liquids. However, it will generally bedesirable to administer them to the skin as compositions orformulations, in combination with a dermatologically acceptable carrier,which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, alcohols or glycols or water-alcohol/glycolblends, in which the present compounds can be dissolved or dispersed ateffective levels, optionally with the aid of non-toxic surfactants.Adjuvants such as fragrances and additional antimicrobial agents can beadded to optimize the properties for a given use. The resultant liquidcompositions can be applied from absorbent pads, used to impregnatebandages and other dressings, or sprayed onto the affected area usingpump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

Examples of useful dermatological compositions which can be used todeliver NAC and/or melatonin to the skin are known to the art; forexample, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat.No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman(U.S. Pat. No. 4,820,508).

Useful dosages of NAC and/or melatonin can be determined by comparingtheir in vitro activity, and in vivo activity in animal models. Methodsfor the extrapolation of effective dosages in mice, and other animals,to humans are known to the art; for example, see U.S. Pat. No.4,938,949.

Generally, the concentration of NAC and/or melatonin in a liquidcomposition, such as a lotion, will be from about 0.1-25 wt-%,preferably from about 0.5-10 wt-%. The concentration in a semi-solid orsolid composition such as a gel or a powder will be about 0.1-5 wt-%,preferably about 0.5-2.5 wt-%.

The amount/preferred dose of NAC, melatonin, an active salt orderivative thereof, or a combination thereof, required for use intreatment will vary not only with the particular salt/compositionselected, but also with the route of administration, the nature of thecondition being treated and the age, weight and condition of thepatient. Importantly, the quantity of NAC, melatonin, an active salt orderivative thereof, or a combination thereof, used should be sufficientto prevent, inhibit, reduce the risk of, or treat cancer and/or prevent,inhibit, reduce the risk of, or treat neurodegeneration. Thus, a varietyof clinical factors will influence the preferred dosage ranges and willbe ultimately at the discretion of the attendant physician or clinician.

In general, however, a suitable dose of NAC will typically be in therange of from about 0.5 to about 10 mg/kg, e.g., from about 2 to about10 mg/kg of body weight per day, preferably in the range of 5 to 9mg/kg/day.

NAC can be conveniently administered in unit dosage form; for example,containing 5 to 1000 mg, conveniently 10 to 750 mg, most conveniently,50 to 500 mg of active ingredient per unit dosage form.

In general, however, a suitable dose of melatonin will typically be inthe range of from about 0.01 to about 0.2 mg/kg, e.g., from about 0.1 toabout 0.2 mg/kg of body weight per day, preferably in the range of 0.05to 0.15 mg/kg/day.

Melatonin can be conveniently administered in unit dosage form; forexample, containing 1 to 20 mg, conveniently 2 to 15 mg, mostconveniently, 3 to 10 mg of active ingredient per unit dosage form.

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations; such as multiple inhalations from an insufflator or byapplication of a plurality of drops into the eye.

Methods for Using NAC and/or Melatonin

Through research of the underlying mechanisms of the formation of cancerand the development of neurodegenerative disease or disorders, Applicanthas made the unexpected discovery of a unifying molecular mechanism ofinitiation for many cancers and neurodegenerative diseases. Thisunifying mechanism involves the oxidation of the carcinogenic 4-hydroxycatechol estrogens (CE) of estrone (E1) and estradiol (E2) to catecholestrogen-3,4-quinones (CE-3,4-Q) resulting in electrophilicintermediates that covalently bind to DNA to form depurinating adductsat the N-7 of guanine and N-3 of adenine by 1,4-Michael addition. Italso involves the oxidation of the catecholamine dopamine and themetabolite catechol (1,2-dihydroxybenzene) of the leukemogenic benzeneto their quinones which react with DNA to form predominantly analogousdepurinating adducts. The resultant apurinic sites in critical genes cangenerate mutations that may initiate various human cancers and/orneurodegenerative diseases or disorders.

Applicant has discovered that NAC and/or melatonin, both of which maycross the blood-brain barrier, are two agents that reduce the formationof such above-mentioned endogenous quinones. It has also been discoveredthat NAC and/or melatonin reduce the formation of depurinating adductsdue to the action of quinones. As such, administration of NAC ormelatonin alone or in combination surprisingly and unexpectantly offersa method for preventing and/or reducing the risk of cancer and/orneurodegenerative diseases.

In a preferred method, compositions comprising NAC and/or melatonin areused for the prevention, inhibition, and/or treatment of cancers such asprimary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer,liver cancer, brain cancer, non-Hodgkin's lymphoma, Hodgkins lymphoma,leukemias, uterine cancer, cervical cancer, bladder cancer, rectalcancer, kidney cancer, colon cancer, and adenocarcinomas such as breastcancer, prostate cancer, ovarian cancer, and pancreatic cancer.

In another preferred method, compositions comprising NAC and/ormelatonin are used for the prevention, inhibition and/or treatment ofneurodegenerative diseases such as diseases associated with the loss ofneurons in different regions of the central nervous system (CNS),including, for example, brain tissue and the spinal cord, such asAlzheimer's disease, amyotrophic lateral sclerosis (“ALS” or “LouGehrig's disease”), Parkinson's disease, Huntington's disease, brainaging, Friedreich's ataxia, multiple sclerosis, diabetic necrosis,ischaemia, and stroke.

Other Agents

The unifying mechanism that has been discovered also suggests otheragents that will be useful for treating or preventing cancer andneurodegenerative diseases. For example, it has been determined thatagents that induce the protective enzyme quinone reductase, whichreduces catechol estrogen quinones to catechol estrogens, will alsoprovide beneficial effects. Two such agents are lipoic acid andresveratrol. Additionally, agents that inhibit CYP1B1 will also providea beneficial effect.

Lipoic acid (1,2-dithiolane-3-pentanoic acid) is an antioxidant becausethe dithiolane structure is a strained five-membered ring that is highlyreactive. The relatively high energy content of the disulfide group inlipoic acid makes it reactive with oxidizing molecules. The reduced formof lipoic acid, dihydrolipoic acid (the two forms are in equilibrium)has greater antioxidant activity. Thus, the potent reducing capacity ofdihydrolipoic acid and the high reactivity of the disulfide groups inlipoic acid make this couple important as an antioxidant defense systemin the cell. The LA/DHLA exhibits free radical (superoxide anion radicaland hydroxyl radicals) scavenging properties, reducing oxidative stress.Because it is a strong reductant, it can regenerate vitamin C, Vitamin Eand GSH from their oxidized forms. Lipoic acid readily crosses theblood-brain barrier. Finally, it is believed that lipoic acid willinduces the protective enzyme quinone reductase, which reduces CEQ backto CE.

Resveratrol, a polyphenolic phytoalexin, is a natural fungicide in morethan 70 plant species. It is an antioxidant and an antimutagen.Resveratrol scavenges hydroxyl radicals, superoxide anion radicals andmetal-induced radicals, thus protecting against lipid peroxidation. Itinhibits cytochrome P450 1A1, 1B1 and 3A4, thus reducing oxidation ofestrogens to catechol estrogens (CE). It is also an inducer of quinonereductase, thus increasing the reduction of CEQ to CE. Resveratrolinhibits dioxin-induced expression of P450 1A1 and 1B1, as well asCE-mediated oxidative damage to DNA in cultured human mammary epithelialcells. Resveratrol also inhibits CYP1B1, the major enzyme that catalyzesformation of 4-catechol estrogens in extrahepatic tissues (like thebreast, prostate, etc. Inhibition of CYP1B1 activity in the breast,would be expected to lower amounts of 4-CE, lower mounts ofCE-3,4-quinones and reduce formation of depurinating 4-CE-DNA adductsthat generate mutations leading to the initiation of cancer.

Accordingly, the compositions of the invention can optionally compriseone or more agents that induce quinone reductase (e.g. lipoic acid andresveratrol). The compositions of the invention can also optionallycomprise one or more agents that inhibit CYP1B1 (e.g. resveratrol).Additionally, the methods of the invention can optionally compriseadministering one or more agents that induce quinone reductase. Themethods of the invention can also optionally comprise administering oneor more agents that inhibit CYP1B1.

Method for Detecting and/or Diagnosing Cancer and/or NeurodegenerativeDisease

As described herein below, 4-CE were 3.5 times more abundant than the2-CE and were 4 times higher than in women without breast cancer,demonstrating that the amount of 4-CE present in a physiological samplecorrelates with cancer. Additionally, a lower level of methylation wasobserved for the CE cancer cases compared to controls. Also, CE-Qconjugate levels were 3 times higher in women with cancer than controls.Therefore, the determination of the presence and/or amount of dopaminequinone, 4-CE, 2-CE, methylation of CE and/or CE-Q conjugates may beuseful in the diagnosis, treatment and/or monitoring of the progressionor remission of cancer, such as breast cancer, and/or neurodegenerativediseases. Thus, there is provided a method for determining the risk ofdeveloping cancer and/or a neurodegenerative disease in a mammalcomprised of determining the amount of endogenous quinone, dopaminequinone, 4-CE, 2-CE, methylation of CE and/or CE-Q conjugates present ina physiological sample from a mammal, such as blood, urine or other bodyfluid or a tissue biopsy, and comparing the determined amount to anamount present in a control sample, wherein an increase in amount ofquinone correlates with cancer, such as breast cancer, and/orneurodegenerative disease. The presence or quantity of quinone in thesample can be determined using any suitable analytical method, such asIR, UV, NMR, Mass Spec or HPLC. A preferred method for detecting ordetermining the presence or amount of estrogen metabolites, dopaminemetabolites, conjugates and depurinating DNA adducts, including 4-CE,2-CE, methylation of CE and/or CE-Q conjugates, is by HPLC withelectrochemical detection.

Screening Method for Identifying New Therapeutic Agents

The present invention also provides a screening method to identify newtherapeutic agents that inhibit the production and/or the activity(e.g., the ability to associate with or bind DNA and form depurinatingDNA adducts) of endogenous quinones. Preferably, the quinones haveformed endogenously from the oxidation of the carcinogenic 4-hydroxycatechol estrogens (CE) of estrone (E₁) and estradiol (E₂) to catecholestrogen-3,4-quinones (CE-3,4-Q) resulting in electrophilicintermediates that covalently bind to DNA to form depurinating adductsat the N-7 of guanine and N-3 of adenine by 1,4-Michael addition. Theresultant apurinic sites in critical genes can generate mutations thatmay initiate various human cancers. Also, the quinones may formendogenously from the oxidation of the catecholamine dopamine and themetabolite catechol (1,2-dihydroxybenzene) of the leukemogenic benzeneto their quinones which can react with DNA to form predominantlyanalogous depurinating adducts. In the case of depurinating adductsresulting from oxidization of dopamine to its quinone, the resultantapurinic sites in critical genes can generate mutations that mayinitiate brain cancer and/or neurodegenerative diseases.

The present invention provides an in vitro binding assay comprisingincubating DNA and catechol estrogen quinones or catecholamine dopaminequinones (e.g., for about 2 hours at 37° C.) with and without acandidate agent. After the incubation period, stable adducts arequantified (e.g., by a ³²P-postlabeling method, as used and describedhereinbelow, and the presence or quantity of depurinating adducts isanalyzed (e.g., by high pressure liquid chromatography, HPLC). Thecomponents of the reaction mixture with the candidate agent are comparedto those of the reaction mixture without the candidate agent (control)to determine whether the candidate agent is able to inhibit or preventthe association/binding of DNA and quinones and/or the formation ofdepurinating adducts. If the quantity of DNA/quinone complexes and/ordepurinating adducts formed in the reaction mixture with the candidateagent is less than the mixture without the candidate agent, then thecandidate agent may be useful in a method for preventing or reducing therisk of cancer and/or neurodegenerative disease.

One cellular screening method comprises contacting a culture of cells(e.g., mammalian cells) with E₂ , contacting a duplicate culture ofcells with a candidate agent and E₂, and measuring the effect of thecandidate agent on the production of CE-3,4-Q, or depurinating adducts.This screening method can identify agents which block the production ofendogenous quinones and/or detoxify them (rendering them unable toproduce depurinating adducts).

Another cell model for screening for therapeutic agents comprisescontacting tissue cultured cells, such as cancerous tissue (e.g.,breast), which has been tested to contain relatively higher than normalamounts of 4-CE or CE-3,4-Q (as determined by analytical methods) with acandidate agent and measuring the effect of the candidate agent on theproduction of 4-CE, CE-3,4-Q or depurinating adducts. If the amount of4-CE, CE-3,4-Q, or depurinating adducts is reduced in the treated sampleas compared to a control sample, the candidate agent may be useful in amethod for preventing or reducing the risk of cancer and/orneurodegenerative diseases.

According to the methods of the invention, a sample can be compared toan appropriate control (e.g., a control mammal or a control cell) thecriteria for selecting an appropriate control are well understood bythose of skill in the art. For example, a control mammal may be asimilar mammal lacking the condition for which you are testing (e.g.,cancer (e.g., breast cancer) or neurodegenerative disease).

The compositions and methods of the invention will now be illustrated bythe following non-limiting Examples.

EXAMPLE I

Initiation of Cancer and Other Diseases by Catechol Ortho-Quinones: AUnifying Mechanism

Introduction

One of the major obstacles in cancer research is that cancer is aproblem of 200 diseases. This viewpoint has impeded researchers fromlooking at the etiology of cancers because the search would beprohibitively complex. For this reason, the etiology of breast, prostateand other human cancers remains virtually unknown. While the expressionof various cancers coincides with the above concept, some scientistsconsider there to be a common, but not yet elucidated, origin for manyprevalent types of cancer.

There is widespread agreement in the scientific community that cancer isbasically a genetic disease—not in the sense that most cancers areinherited, but in the sense that cancer is triggered by geneticmutations. Thus, cancer can be considered a disease of mutated criticalgenes that modulate cell growth and death. These include oncogenes andtumor suppressor genes, which give rise to transformation and abnormalcell proliferation. Understanding the origin of these mutations opensthe door to strategies for controlling and preventing cancer.(Chakravarti D. et al., Mutation Res., 456, 17-32 (2000) and Oncogene,20:7945-7953 (2001); Weinberg R. A., Sci. Am., 275, 62-77 (1996).)

A second barrier to the progress of cancer research is related to thereluctance of the scientific community to recognize that the naturalestrogens, including estrone (E₁) and estradiol (E₂), are truecarcinogens, which induce tumors in various hormone-dependent andindependent organs of several animal species and strains. (InternationalAgency for Research on Cancer Monographs, 6, 99-132 (1974);International Agency for Research on Cancer Monographs, 21, 279-362(1979); International Agency for Research on Cancer Monographs, Anupdating of LARC monographs volumes 1 to 42 (1987); IARC Monographs,Suppl. 7, 272-310).)

A third obstacle to the progress of research on breast and otherhormone-dependent cancers is related to the standard paradigm, stated byFeigelson and Henderson, that estrogens, through receptor-mediatedprocesses, “affect the rate of cell division and, thus, manifest theireffect on the risk of breast cancer by causing proliferation of breastepithelial cells. Proliferating cells are susceptible to genetic errorsduring DNA replication, which, if uncorrected, can ultimately lead to amalignant phenotype”. While there is no doubt that estrogen-mediatedcontrol of cell proliferation plays a role in the development of breastand other hormone-dependent cancers, accumulating evidence suggests thatspecific oxidative metabolites of estrogens, if formed, can be theendogenous ultimate carcinogens. By reacting with DNA, they cause themutations leading to cancer. This initiating mechanism occurs inhormone-dependent and independent tissues. (Feigelson H. S. andHenderson B. E., Carcinogenesis, 17: 2279-2284 (1996); INCI Monograph27, E. Cavalieri and E. Rogan (eds.), Oxford Press, Washington (2000).)

Abbreviations

Ade, adenine; BP, benzo[a]pyrene, CE, catechol estrogen(s); CE-Q,catechol estrogen quinone(s); CE-SQ, catechol estrogen semiquinone(s);COMT, catechol-O-methyltransferase; CYP, cytochrome P450; DB[a,l]P,dibenzo[a,l]pyrene; anti-DB[a,l]PDE,anti-dibenzo[a,l]pyrene-11,12-dihydrodiol-13,14-epoxide; DMBA,7,12-dimethylbenz[a] anthracene; E₁, estrone; E₂, estradiol; Gua,guanine; GSH, glutathione; H, Harvey; OHE₂, hydroxyestradiol; PAH,polycyclic aromatic hydrocarbon(s); PCR-RFLP, polymerase chainreaction-restriction fragment length polymorphism(s); TDG, T.G-DNAglycosylase.

Results and Discussion

Covalent Binding of Carcinogens to DNA: Stable and Depurinating Adducts

Chemical carcinogens covalently bind to DNA to form two types of DNAadducts: stable ones that remain in DNA unless removed by repair anddepurinating ones that are released from DNA by destabilization of theglycosyl bond (FIG. 1). Stable adducts are formed when carcinogens reactwith the exocyclic N⁶ amino group of adenine (Ade) or N² amino group ofguanine (Gua), whereas depurinating adducts are obtained whencarcinogens covalently bind at the N-3 or N-7 of Ade or the N-7 orsometimes C-8 of Gua. The loss of Ade or Gua by depurination leads toformation of apurinic sites that can generate the mutations leading totumor initiation. (Cavalieri E. L. and Rogan E. G., Pharmacol. Ther.,55, 183-99 (1992); Cavalieri E. L. and Rogan E. G., Mechanisms of tumorinitiation by polycyclic aromatic hydrocarbons in mammals, In: TheHandbook of Environmental Chemistry: PAHs and Related Compounds, 3J, pp.81-117, Neilson A. H. (ed.), Springer, Heidelberg, Germany (1998).)

Identification and quantification of polycyclic aromatic hydrocarbon(PAH)-DNA adducts led to the discovery that there is a correlationbetween depurinating adducts and oncogenic mutations, suggesting thatthese adducts are the primary culprits in the tumor initiating pathway.This discovery was made by identifying the DNA adducts formed in mouseskin by dibenzo[a,l]pyrene (DB[a,l]P), 7,12-dimethylbenz[a]anthracene(DMBA) and benzo[a]pyrene (BP) and, at the same time, determining themutations in the Harvey (H)-ras oncogene in mouse skin papillomasinitiated by these three PAH (Table 1). TABLE 1 Correlation ofdepurinating adducts with H-ras mutations in mouse skin papillomas H-rasMutations No. of mutations PAH Major DNA Adducts No. of mice codon DMBAN7Ade (79%)  4/4 CAA→CTA 61 DB[a, l]P N7Ade (32%) 10/12 CAA→CTA 61 N3Ade(49%) BP C8Gua + N7Gua (46%) 10/20 GGC→GTC 13 N7Ade (25%)  5/20 CAA→CTA61

These mutations correlate with the predominant formation of depurinatingAde adducts by DMBA and DP[a,l]P and the two-to-one ratio ofdepurinating Gua to Ade adducts formed by BP. This pattern of rasmutations suggests that the oncogenic mutations in mouse skin papillomasinduced by these PAH are generated by misrepair of the apurinic sitesderived from loss of the depurinating adducts. Because thousands ofapurinic sites are formed by cells each day, repair of apurinic sitesinduced by PAH might be expected. The level of apurinic sites arisingfrom treatment with PAH is, however, 15-120 times higher than thoseformed spontaneously, suggesting that this large increase in apurinicsites could lead to misrepair. In summary, apurinic sites can generatethe mutations that play a critical role in the initiation of cancer, andformation of depurinating adducts has become the common denominator forrecognizing the potential of a chemical to initiate cancer. (CavalieriE. L. and Rogan E. G., Mechanisms of tumor initiation by polycyclicaromatic hydrocarbons in mammals. In: The Handbook of EnvironmentalChemistry: PAHs and Related Compounds, 3J, pp. 81-117, Neilson A. H.(ed.), Springer, Heidelberg, Germany (1998); Chakravarti D. et al.,Proc. Natl. Acad. Sci. USA, 92, 10422-10426 (1995); Chakravarti D. etal., Mutation Res., 456, 17-32 (2000); Lindahl T. and Nyberg B.,Biochemistry, 11; 3610-3618 (1972); Chakravarti D. et al., Oncogene, 16,3203-3210 (1998).)

Formation, Metabolism and DNA Adducts of Estrogens

Evidence that depurinating PAH-DNA adducts play a major role in tumorinitiation provided the impetus for discovering the estrogen metabolitesthat form depurinating DNA adducts and can be potential endogenousinitiators of cancer. Catechol estrogens (CE) are among the majormetabolites of E₁ and E₂. If these metabolites are oxidized to theelectrophilic CE quinones (CE-Q), they may react with DNA. Specifically,the carcinogenic 4-CE are oxidized to CE-3,4-Q, which react with DNA toform depurinating adducts. These adducts generate apurinic sites thatmay lead to oncogenic mutations, thereby initiating cancer. (CavalieriE. L. and Rogan E. G., Pharmacol. Ther., 55; 183-99 (1992); Cavalieri E.L. and Rogan E. G. Mechanisms of tumor initiation by polycyclic aromatichydrocarbons in mammals. In: The Handbook of Environmental Chemistry:PAHs and Related Compounds, 3J, 81-117, Neilson A. H. (ed.), Springer,Heidelberg, Germany (1998); Chakravarti D. et al., Proc. Natl. Acad.Sci. USA, 92, 10422-10426 (1995); Cavalieri, et al., Proc. Natl. Acad.Sci. USA, 94, 10937-10942 (1997); Liehr J. G., et al., J. SteroidBiochem., 24, 353-356 (1986); Li J. J. and Li S. A., Fed. Proc.. 46,1858-1863 (1987); Newbold R. R. and Liehr J. G., Cancer Res., 60,235-237 (2000); Li K. M., et al., Proc. Am. Assoc. Cancer Res., 39, 636(1998); Chakravarti D., et al., Mutation Res., 456, 17-32 (2000);Chakravarti D., et al., Oncogene, 16, 3203-3210 (1998); Chakravarti D.,et al., Oncogene, 20, 7945-7953 (2001).)

Estrogen Metabolism.

E₁ and E₂ are obtained by aromatization of 4-androsten-3,17-dione andtestosterone, respectively, catalyzed by cytochrome P450 (CYP)19,aromatase (FIG. 2). The estrogens E₁ and E₂ are biochemicallyinterconvertible by the enzyme 17β-estradiol dehydrogenase. E₁ and E₂are metabolized via two major pathways: formation of CE and, to alesser, extent, 16α-hydroxylation (not shown in FIG. 2). The CE formedare the 2- and 4-hydroxylated estrogens. The major 4-hydroxylase inextrahepatic tissues is CYP 1B1. In general, the CE are inactivated byconjugating reactions such as glucuronidation and sulfation, especiallyin the liver (not shown in FIG. 2). The most common pathway ofconjugation in extrahepatic tissues, however, occurs by O-methylationcatalyzed by the ubiquitous catechol-O-methyltransferase (COMT). (SpinkD. C., et al., J. Steroid Biochem. Mol. Biol., 51, 251-258 (1994); HayesC. L., et al., Proc. Natl. Acad. Sci. USA, 93, 9776-9781 (1996); SpinkD. C., et al., Carcinogenesis, 19, 291-298 (1998); Männistö P. T. andKaakola S., Pharmacol. Rev., 51, 593-628 (1999).)

A reaction that is competitive with the conjugation of CE is theircatalytic oxidation to CE-semiquinones (CE-SQ) and CE-Q (FIG. 2). CE-SQand CE-Q can be neutralized by conjugation with glutathione (GSH). Asecond inactivating pathway for CE-Q is their reduction to CE by quinonereductase and/or cytochrome P450 reductase. If these two inactivatingprocesses are insufficient, CE-Q may react with DNA to form stable anddepurinating adducts (FIG. 2). The carcinogenic 4-CE are oxidized toform predominantly the depurinating adducts 4-OHE₁(E₂)-1-N3Ade and4-OHE₁(E₂)-1-N7Gua. Carcinogenic 2-CE are oxidized to form predominantlystable adducts, 2-OHE₁(E₂)-6-N⁶dA and 2-OHE₁(E₂)-6-N²dG, but alsodepurinating adducts to a much lesser extent. (DT Diaphorase A quinonereductase with special functions in cell metabolism and detoxification(Emester L, Estabrook R W, Hochstein P, Orrenius S., Eds.) ChemicaScripta 27A (1987); Roy, D. and Liehr J. G., J. Biol. Chem., 263,3646-3651 (1988); Cavalieri E., et al., Estrogens as endogenousgenotoxic agents: DNA adducts and mutations. In: JNCI Monograph 27:Estrogens as Endogenous Carcinogens in the Breast and Prostate, pp.75-93, E. Cavalieri and E. Rogan (eds.), Oxford Press, Washington(2000); Liehr J. G., et al., J. Steroid Biochem., 24, 353-356 (1986); LiJ. J. and Li S., Fed. Proc., 46, 1858-1863 (1987); Newbold R. R. andLiehr J. G., Cancer Res., 60, 235-237 (2000); Cavalieri E. L., et al.,Proc. Natl. Acad. Sci. USA, 94, 10937-10942 (1997); Li K. M., et al.,Proc. Am. Assoc. Cancer Res., 39, 636 (1998), Stack D. E., et al., Chem.Res. Toxicol., 9, 851-859 (1996); Dwivedy I., et al., Chem. Res.Toxicol., 5, 828-833 (1992); Van Aerden C., et al., Analyst, 123,2677-2680 (1998).)

Redox Cycling of Catechol Estrogen Semiquinones and Quinones.

Redox cycling (FIGS. 2 and 3) generated by reduction of CE-Q to CE-SQ,catalyzed by cytochrome P450 reductase, and subsequent oxidation back toCE-Q by molecular oxygen forms superoxide anion radicals (O₂.⁻). TheseO₂.⁻ dismutate to H₂O₂, either spontaneously or, even faster, when thereaction is catalyzed by superoxide dismutase. H₂O₂ is rathernonreactive, except in the presence of reduced transition metal ions,namely Fe²⁺ and Cu⁺, which cause formation of indiscriminate oxidants,the hydroxyl radicals. These reactive species can damage DNA byformation of oxygenated bases. Concurrently, hydroxyl radicals caninitiate the lipid peroxidation process, generating lipid hydroperoxidesthat can serve as unregulated cofactors for oxidation of CE bycytochrome P450. In contrast, under normal conditions nicotinamidedinucleotide phosphate NADPH serves not only as a cofactor, but alsoregulates cytochrome P450 in the oxidation of CE. Thus, once lipidhydroperoxides are formed, the oxidation of CE to CE-SQ and CE-Q canbecome a self-generating process that unbalances estrogen homeostasisand leads to formation of CE-Q. (Liehr J. G. and Roy D., Free RadicalBiol. Med., 8, 415-423 (1990); Nutter L. M., et al., Chem. Res.Toxicol., 7, 23-28 (1994); Malins D. C., et al., Cancer, 17, 3036-3043(1993); Lavigne J. A., et al., Cancer Res., 61, 7488-7494 (2001); KappusH., Lipid peroxidation: Mechanisms, analysis, enzymology and biologicalrelevance. In: Sies, H., ed., Oxidative Stress, New York, AcademicPress, 273-310 (1985).)

Binding of Catechol Estrogen Quinones to DNA.

To determine whether DNA adducts are formed in biological systems,E₂-3,4-Q or enzymatically-activated 4-hydroxyestradiol (4-OHE₂) wasreacted with DNA for 2 h at 37° C. The stable adducts were quantified bythe ³²P-postlabeling method, and the depurinating adducts were analyzedby high pressure liquid chromatography (HPLC) interfaced with anelectrochemical detector. When E₂-3,4-Q reacted with DNA, almost thesame amount of the depurinating adducts 4-OHE₂-1-N3Ade and4-OHE₂-1-N7Gua were obtained, and the amount of stable adducts was 0.02%of the depurinating ones. Activation of 4-OHE₂ by horseradish peroxidasegave similar results, whereas the mammalian lactoperoxidase produced asimilar amount of N3Ade adduct, but about 50% more N7Gua adduct. Thesame two depurinating adducts were obtained in equal but smaller amountswhen 4-OHE₂ was activated with tyrosinase or phenobarbital-induced ratliver microsomes. In all cases, the level of stable adducts was 0.02% orless compared to the depurinating adducts (Li K. M., et al., Proc. Am.Assoc. Cancer Res., 39, 636 (1998); Cavalieri, et al., unpublishedresults).

DNA adducts were analyzed in vivo in rat mammary gland and mouse skinafter treatment of the animals with E₂-3,4-Q or 4-OHE₂. Female ACI rats,which are susceptible to E₂-induced mammary tumors, were treated byintramammillary injection of E₂-3,4-Q or 4-OHE₂ (200 nmol in 20 μLDMSO/gland at four teats) for 1 h. The mammary tissue was excised,extracted and analyzed for stable and depurinating adducts. N3Ade andN7Gua adducts from both 4-OHE₂ and 4-OHE, were detected in the range of100-300 μmol/mol DNA-P. The level of stable adducts was not above thelow level detected in untreated mammary tissue. Similarly, female SENCARmice were treated topically on a shaved area of dorsal skin withE₂-3,4-Q [200 nmol in 50 μL acetone/DMSO (9:1)] for 1 h. The treatedarea of skin was excised, extracted and analyzed for stable anddepurinating adducts. Equal amounts of 4-OHE₂-1-N3Ade and4-OHE₂-1-N7Gua, approximately 12 μmol/mol DNA-P, were detected, and theamount of stable adducts was 0.02% of the depurinating adducts. Theseresults in rats and mice demonstrate that the depurinating CE-DNAadducts are formed in vivo, generating apurinic sites in the DNA thatcould lead to oncogenic mutations. (Shull J. D., et al., Carcinogenesis,18, 1595-1601 (1997); Cavalieri, et al., unpublished results;Chakravarti D., et al., Oncogene, 20, 7945-7953 (2001).)

Depurinating Adducts and Induction of Mutations

Mouse skin provides a model system to study the conversion of DNAlesions, such as carcinogen-induced depurinating and stable DNA adducts,into mutations. In mouse skin, tumor initiation occurs when these DNAlesions are converted into oncogenic mutations in the H-ras gene.Previous studies indicated that stable adducts are inefficiently removedby excision repair, and cells containing these adducts enter theS-phase. In the S-phase, occasional mutations are induced whenreplicative DNA polymerases go over adducted templates. Therefore, itwas concluded that adduct-induced mutagenesis occurs in proliferatingcells. These studies, however, did not address the fate of apurinicsites formed by the depurinating adducts. (Maher V. M., and McCormick J.J., Role of DNA lesions and repair in the transformation of human cells.In: D. Grunberger, S. P. Groff (eds) Mechanisms of CellularTransformation by Carcinogenic Agents. Pergamon Press, New York, 135-149(1987); Kaufman W. K., Cancer Metastasis Rev, 14, 31-41 (1995); MoriyaM., et al., Biochemistry, 35, 16646-16651 (1996).)

Resting Cells are Greatly Susceptible to Tumor Formation.

Mouse skin is most susceptible to tumor formation by carcinogens duringthe telogen phase of the hair cycle. At telogen, epidermal thickness islow, indicating that at these times epidermal cells are in the restingphase. DMBA, which forms 99% depurinating adducts and 1% stable adducts,induces several-fold more tumors when applied to resting phase skin.This suggests that apurinic sites induced by the depurinating DNAadducts may be most efficiently converted into oncogene-activatingmutations in G0-G1 phase cells. These questions were examined withDB[a,l]P, the strongest among PAH carcinogens, which also forms 99%depurinating adducts and 1% stable adducts in mouse skin DNA and inducesthe H-ras codon 61 (CAA to CTA) mutation in tumors. (Devanesan P. D., etal., Chem. Res. Toxicol., 6, 364-371 (1993); Andreasen E., Acta. Pathol.Scand., 32, 157-164 (1953); Cavalieri E. L. and Rogan E. G., Mechanismsof tumor initiation by polycyclic aromatic hydrocarbons in mammals. In:The Handbook of Environmental Chemistry: PAHs and Related Compounds, 3J,81-117, Neilson A. H. (ed.), Springer, Heidelberg, Germany (1988);Chakravarti D., et al., Proc. Natl. Acad. Sci. USA, 92, 10422-10426(1995).)

Using a polymerase chain reaction-restriction fragment lengthpolymorphism (PCR-RFLP) technique, it was found that treatment of mouseskin with 200 nmol of DB[a,l]P resulted in the induction of these codon61 mutations as early as one day after treatment. In this technique, asegment of the H-ras gene is PCR-amplified and the product is restrictedwith XbaI to examine the induction of a RFLP from the codon 61 mutation.It was observed that at one day, 0.1% of the H-ras genes in the treatedarea of skin contained the codon 61 mutation, and then the population ofthese mutations increased to a maximum of 5% between 3 and 4 days afterDB[a,l]P treatment. Subsequently, the level of mutations was reduced tobackground levels (0.0001%). The early time of induction of the codon 61mutations (one day after DB[a,l]P treatment) coincides with suppressionof DNA synthesis and induction of excision repair. Therefore, perhapsDB[a,l]P-induced DNA damage is converted into mutations by error-proneexcision repair in pre-S-phase cells. (Chakravarti D., et al., Oncogene,16, 3203-3210 (1998); Slaga T. J., et al., Cancer Res., 34a, 771-777(1974); Sawyer T. W., et al., Carcinogenesis, 9, 1197-1202 (1988); GillR. D., et al., Environ. Mol. Mutagen., 18, 200-206 (1991).)

Error-Prone Repair of Apurinic Sites is a Mechanism of Tumor Initiation.

Evidence in support of this was obtained from a comparative study ofmutations in the mouse skin H-ras gene induced by 200 nmol of DB[a,l]Por 200 nmol of anti-DB[a,l]P-11,12-dihydrodiol-13,14-epoxide(anti-DB[a,l]PDE). Unlike DB[a,l]P, anti-DB[a,l]PDE forms 97% stableadducts and 3% depurinating adducts in DNA. In these experiments, thetypes of mutations that are induced in the early preneoplastic times wasidentified (12 h to 9 days after treatment) and then analyzed whetherthese mutations were induced as a result of error-prone repair (FIG. 4).The mutations were identified by PCR amplifying a segment of the H-rasgene from DNA extracted from mouse skin treated with one of thecarcinogens, cloning the PCR products in a plasmid, isolating individualsubclones and sequencing the H-ras inserts to identify mutations.(Chakravarti D., et al., Mutation Res., 456, 17-32 (2000); Cavalieri E.L. and Rogan E. G., Mechanisms of tumor initiation by polycyclicaromatic hydrocarbons in mammals. In: The Handbook of EnvironmentalChemistry: PAHs and Related Compounds, 3J, 81-117, Neilson A. H. (ed.),Springer, Heidelberg, Germany (1998).)

The mutation spectra induced by DB[a,l]P contained 90% A/T to G/Cmutations at day 1. This correlated with the abundant DB[a,l]P-Adedepurinating adducts (81% of total adducts) and suggested that these A/Tto G/C mutations were induced at Ade depurinations. Thus, the adductscould be correlated with these early preneoplastic mutations, as well aswith the clonal H-ras mutations found in the tumors (Table 1). If Adedepurinations induce these A/T to G/C mutations, they may be A to Gmutations generated as G.T heteroduplexes by error-prone excision repair(FIG. 5).

Using a novel technique, it was determined that these A to G mutationsin the H-ras gene are initially induced as G.T heteroduplexes. In thistechnique, G.T heteroduplexes in skin DNA are converted toG.apyrimidinic sites by treatment with T.G-DNA glycosylase (TDG) (FIG.5). (Chakravarti D., et al., Mutation Res., 456, 17-32 (2000).)

Depurinated DNA templates are refractory to PCR amplification. Todemonstrate this point, a mixture of two plasmids (one contained thewild type H-ras exon 1-2 segment (pWT) and the other contained the sameDNA with the codon 61 (CAA to CTA) mutation (pMUTX)) was PCR amplified.The yield of pMUTX in the PCR product was determined by XbaI digestion.When pMUTX was incubated in an acidic buffer to induce a relativelysmall amount of depurination that did not significantly degrade theplasmid (˜1 depurination/H-ras segment), mixed with untreated pWT andPCR amplified, a drastically reduced amount of the product wasXbaI-digestible. This confirmed that depurinated templates arerefractory to PCR amplification. Failure to score mutations in pWTdepurinated either by acid-treatment (FIG. 4A) or through depurinatingadduct formation by E₂-3,4-Q (FIG. 7A) may be related to theunavailability of depurinated templates for PCR amplification.(Chakravarti D., et al., Mutation Res., 456, 17-32 (2000); Fromenty B.,et al., Nucl. Acids Res., 28, e50 (2000).)

Since a basic site-containing DNA molecules are refractory to PCRamplification, the conversion of G.T heteroduplexes into G. apyrimidinicsites makes H-ras molecules containing these heteroduplexesunamplifiable. Under these circumstances, PCR preferentially amplifiestemplates that do not contain G.T heteroduplexes. As a result, PCRamplification of the H-ras gene from TDG-treated skin DNA, followed bycloning the PCR product and isolating and determining the sequence ofindividual subclones, causes a specific, drastic reduction of A to Gmutations in the mutation spectra. In addition, the preferential PCRamplification artificially enriches low-abundance mutations that areobserved only in TDG-treated spectra.

Following the entry of skin cells into S-phase, however, the G.Theteroduplexes are converted into G.C and A.T base pairs by one round ofreplication. At this stage, TDG treatment does not reduce the frequencyof A to G mutations in the spectra. Thus, the specific reduction of A toG mutations in the mutation spectra by the TDG-PCR procedurecharacterizes these mutations as G.T heteroduplexes. The TDG-PCRprocedure resulted in a drastic reduction in the population of A/T toG/C mutations on day 1, but did not make a significant change at days 2and 3 (Table 2). Therefore, A to G mutations remained as G.Theteroduplexes until one day after DB[a,l]P treatment of the skin;beyond which they were present as G.C and A.T mutations, presumably byreplication. Flow cytometric analysis of epidermal keratinocytesisolated from DB[a,l]P-treated mouse skin confirms that cells begin toenter the S-phase one day after the treatment. (Chakravarti D., et al.,Mutation Res., 456, 17-32 (2000); Chakravarti et al., unpublishedresults).

A major difference in mutation spectra induced by DB[a,l]P andanti-DB[a,l]PDE was the presence or absence of multiple codon 61 (CAA toCTA) mutations in early preneoplastic skin. These mutations weredetectable one day after DB[a,l]P treatment by the PCR-RFLP procedure,which indicated that they constitute 0.1% of H-ras genes, and in themutation spectrum obtained after TDG treatment of skin DNA. Since theseCAA to CTA mutations were observed during the active repair period, itwas hypothesized that they were also induced by error-prone repair asT.T heteroduplexes. Since these mutations were present in days 2-3 in asignificantly greater frequency relative to other mutations in thespectra, it was hypothesized that the increase in frequency was due to aclonal proliferation of codon 61-mutated (initiated) cells. Furtherstudies suggest that at days 2-3, these codon 61-mutated cells expressactivated Ras protein (Chakravarti D., et al., Oncogene, 16, 3203-3210(1998); Chakravarti D., et al., Mutation Res., 456, 17-32 (2000);Chakravarti et al., unpublished observations).

On the other hand, anti-DB[a,l]PDE formed approximately 50% A/T to G/Cmutations, which correlated with 48.5% formation of anti-DB[a,l]PDE-Adestable adducts in mouse skin DNA (Table 2). The frequency of thesemutations was not significantly reduced by the TDG-PCR procedure,indicating that these mutations were not induced by error-prone repair.Studies conducted in other laboratories also indicate that nucleotideexcision repair of bulky stable adducts is error-free. When the pWTplasmid was treated with anti-DB[a,l]PDE in vitro (97% bulky stableadducts) and subjected to PCR amplification, A/T to G/C mutations wasalso found to constitute 50% of all mutations (5 out of 10) (FIG. 4A).These mutations are induced by translesional synthesis over bulky stableadducts by the PCR polymerases. If, as has been proposed by others, onlya small population of PAH-induced bulky stable adducts is removed bypre-replication repair, a large fraction of anti-DB[a,l]PDE-inducedadducts would persist in the mouse skin DNA. It is, therefore, possiblethat the mutations found in anti-DB[a,l]PDE-treated mouse skin DNA areadduct-induced PCR artifacts. The similarity of the frequencies of A/Tto G/C mutations in vitro and in skin is consistent with this idea.TABLE 2 The frequency of changes in DB[a, l]P-induced A/T to G/Cmutations by TDG treatment followed by PCR A/T to B/C mutations/ totalmutations PAH DNA Day −TDG +TDG anti-DB[a, l]PDE pWT —  5/10 (50%)  3/5(60%) skin 1  5/8 (62.5%)  4/5 (80%) DB[a, l]P skin 1 10/11 (90%) 2/10(20%) 2 11/35 (31%) 6/22 (27%) 3  7/22 (31%) 8/25 (32%)(Watanabe M., et al., Mutat Res., 146, 285-294 (1985); Maher V. M. andMcCormick J. J., Role of DNA lesions and repair in the transformation ofhuman cells. In: D. Grunberger, S. P. Groff (eds) Mechanisms of CellularTransformation by Carcinogenic Agents. Pergamon Press, New York, pp.135-149 (1987).)

Four days after treating mouse skin with DB[a,l]P, no codon 61 mutationswere observed in 48 plasmids that contained 15 other mutations (FIG.4B). No definite patterns of mutations were recognized at this time. Atdays 5 and 6, the mutation spectra were mainly limited to codon 52 (CTAto CCA) mutations. This coincided with the early phase ofDB[a,l]P-induced hyperplasia that starts at day 5 and persists beyondday 10. The codon 52 mutation may be oncogenic, but further study isrequired. (Casale, G. P., et al., Fund. Appl. Tox., 36, 71-78 (1997);Casale, G. P., et al., Mol. Car., 27, 125-140 (2000).)

The repair error-induced A/T to G/C mutations in DB[a,l]P-treated mouseskin frequently occurred 3′ to a sequence element, TGN-doublet (FIG. 6),whereas these mutations in anti-DB [a,l]PDE-treated skin did not show asequence context preference (not shown in FIG. 6). This suggests thatthe sequence context of the depurinated base may determine the erroneousbase incorporated by repair. It is also noted that DB[a,l]P inducesapproximately 120-fold more depurinations through the depurinatingadducts than are formed by spontaneous base loss (10,000-20,000depurinations/cell/day). This raised the possibility that abundantdepurination may be a factor in inducing infidelity in repair.(Chakravarti D., et al., Mutation Res., 45, 17-32 (2000); Cavalieri E.L. and Rogan E. G., Mechanisms of tumor initiation by polycyclicaromatic hydrocarbons in mammals. In: The Handbook of EnvironmentalChemistry: PAHs and Related Compounds, 3J, 81-117, Neilson A. H. (ed.),Springer, Heidelberg, Germany (1998); Chakravarti D., et al., Proc.Natl. Acad. Sci. USA, 92, 10422-10426 (1995); Lindahl T. and Nyberg B.,Biochemistry, 11, 3610-3618 (1972); Lindahl T. Nature, 362, 709-715(1993).)

Effect of a Burst of DNA Depurination.

Treatment of mouse skin with E₂-3,4-Q provided evidence that abundantdepurination may induce errors in repair. Like DB[a,l]P, E₂-3,4-Q formspredominantly depurinating adducts in mouse skin DNA, consisting ofroughly equal amounts of two depurinating adducts (4-OHE₂-1-N3Ade and4-OHE₂-1-N7Gua). The N3Ade adduct depurinates instantaneously after itsformation, whereas the N7Gua adduct depurinates slowly, with a half-lifeof 5 h. The difference in the rate of depurination of the two adductsprovided a way to examine the effect of abundant depurination on repairfidelity. Briefly, E₂-3,4-Q would challenge the mouse skin repairmachinery with a burst of Ade-specific depurination and slow-releaseGua-specific depurination. Should a burst of depurination be acontributing factor in causing repair to be error-prone, a greaterfrequency of Ade-specific mutations compared to Gua-specific mutationswould be expected in the mouse skin DNA. (Chakravarti D., et al.,Oncogene, 20, 7945-7953 (2001); Li K.- M., et al., Proc. Am. Assoc.Cancer Res., 40, 46 (1999).) Treatment of mouse skin with 200 nmol ofE₂-3,4-Q induced primarily A/T to G/C mutations in the H-ras gene (FIG.7). For example, 6 h after E₂-3,4-Q treatment, 7 mutations wereidentified among 29 H-ras inserts. Five of the seven were A/T to G/Cmutations. At 12 h, four out of the six mutations found in 30 H-rasinserts were A/T to G/C mutations. At day 1, seven out of the 11mutations found among 50 plasmids were A/T to G/C mutations. Cells donot have enough time to replicate by 6 h, but they may undergo repair.The observation that E₂-3,4-Q induces mutations at 6 h is, therefore, abasis to propose that these mutations are induced by error-prone repair.To confirm this, TDG-PCR analysis of these mutations was conducted (FIG.7). Specifically, at 6 h, TDG treatment reduced the frequency of the A/Tto G/C mutations from 5 in 29 H-ras inserts to 0 in 33 H-ras inserts. At12 h, the change was from 4 in 30 H-ras inserts to 0 in 41 plasmids.These results suggest that at 6-12 h, A/T to G/C mutations were in theform of G.T heteroduplexes. By day 1, a major change in the frequency ofA/T to G/C mutations was not observed, following TDG treatment,suggesting that G.T heteroduplexes were present as G.C and A.T pairs.The TDG-treated 1 day spectrum was dominated by two clonal mutations ofequal frequency (codon 16 AAG to AGG and intronic C to T mutations).Because it is unlikely that the intronic mutation would affect Rasactivity and the two clonal mutations were found in the same frequency,these mutations may be allelic, belonging to a clonally proliferatingpopulation. In contrast, TDG treatment of day 3 DNA did not make anyperceptible changes from the TDG-untreated spectrum. This suggests thatthe mutations found at day 3 were double-stranded and could not beaffected by TDG treatment. (Chakravarti D., et al., Oncogene, 20,7945-7953 (2001).)

E₂-3,4-Q-induced early A/T to G/C mutations were frequently found at Adedepurinations 5′ to G residues. This supports the hypothesis that thesequence context of depurination influences the selection of which baseis incorporated during error-prone repair.

Although these studies suggest that depurinating adducts play a majorrole in inducing transforming mutations to begin the process oftumorigenesis, the stable adducts can also contribute to theseprocesses. Studies indicate that erroneous base incorporation duringreplication over various bulky stable adducts contributes to theinduction of transforming mutations. For example, theBP-7,8-dihydrodiol-9,10-epoxide-N²dG stable adduct induces Aincorporation, forming G to T mutations and the corresponding N⁶dAstable adduct induces C incorporation, forming A to G mutations. Similarstudies indicate that E₂-2,3-Q, which induces primarily bulky stableadducts, is also mutagenic. The 2-OHE₂-N⁶dA stable adducts cause mostlyA to T mutations and some A to G mutations, whereas 2-OHE₂-N²dG stableadducts cause mainly G to T mutations. (Moriya M., et al., Biochemistry,35, 16646-16651 (1996); Chary P., et al., J. Biol. Chem., 270, 4990-5000(1995); Stack D. E., et al., Chem. Res. Toxicol., 9, 851-859 (1996);Dwivedy I., et al., Chem. Res. Toxicol., 5, 828-833 (1992); Van AerdenC., et al., Analyst, 123, 2677-2680 (1998); Terashima I., et al.,Biochemistry, 37, 8803-8807 (1998); Terashima I., et al., Biochemistry,37: 13807-13815 (1998); Terashima I., et al., Biochemistry, 40: 8-14(2001).)

Estrogen Homeostasis

There are several factors that unbalance estrogen homeostasis, namely,the equilibrium between activating and deactivating metabolic pathwayswith the scope of averting the reaction of endogenous CE-Q with DNA(FIG. 2). The first critical factor could be excessive synthesis of E₂by overexpression of aromatase, CYP19, in target tissues and/or thepresence of excess sulfatase that converts stored E₁ sulfate to E₁. Theobservation that breast tissue can synthesize E₂ in situ suggests thatmuch more E₂ is present in some sites of target tissues than would bepredicted from plasma concentrations. (Miller W. R. and O'Neill J.,Steroids, 50, 537-548 (1987); Simpson E. R., et al., Endocrine Rev., 15,342-355 (1994); Yue W., et al., Cancer Res., 58, 927-932 (1998); Yue W.,et al., Cancer, 6, 157-164 (1999); Jefcoate C. R., et al.,Tissue-specific synthesis and oxidative metabolism of estrogens. In:JNCI Monograph 27: Estrogens as Endogenous Carcinogens in the Breast andProstate, pp. 95-112, Cavalieri E. and Rogan E. (eds.), Oxford Press,Washington (2000); Reed M. J. and Purohit A. Endocrine Review, 18,701-715 (1997).)

A second critical factor in unbalancing estrogen homeostasis might bethe presence of high levels of 4-CE due to overexpression of CYP1B1,which converts E₂ predominantly to 4-OHE₂ (FIG. 2). A relatively largeamount of 4-CE could lead to more extensive oxidation to CE-3,4-Q, withincreased likelihood of damaging DNA. (Spink D. C., et al., J. SteroidBiochem. Mol. Biol. 51, 251-258 (1994); Hayes C. L., et al., Proc. Natl.Acad. Sci. USA, 93, 9776-9781 (1996); Spink D. C., et al.,Carcinogenesis, 19, 291-298 (1998).)

A third factor could be a lack or low level of COMT activity. If thisenzyme is insufficient, either through a low level of expression or itslow activity allele, 4-CE will not be effectively methylated,facilitating their oxidation to the ultimate carcinogenic metabolitesCE-3,4-Q (FIG. 2).

Studies in Syrian Golden Hamsters.

The hamster provides an excellent model for studying activation anddeactivation (protection) of estrogen metabolites in relation toformation of CE-Q. In fact, implantation of E₁ or E₂ in male Syriangolden hamsters induces renal carcinomas in 100% of the animals, butdoes not induce liver tumors. Therefore, comparison of the profiles ofestrogen metabolites, conjugates and DNA adducts in the two organsshould provide information concerning the imbalance in estrogenhomeostasis generated by treatment with E₂. Hamsters were injected with8 μmol of E₂ per 100 g body weight, and liver and kidney extracts wereanalyzed for 31 estrogen metabolites, conjugates and depurinating DNAadducts by HPLC interfaced with an electrochemical detector. Neither theliver nor the kidney contained 4-methoxyCE, presumably due to the knowninhibition of COMT by 2-CE. More 0-methylation of 2-CE was observed inthe liver, whereas more formation of CE-Q was detected in the kidney(Table 3). (Li, J. J., et al., Cancer Res., 43, 5200-5204 (1983);Cavalieri E. L., et al., Chem. Res. Toxicol., 14, 1041-1050 (2001); RoyD., et al., Carcinogenesis, 11, 459-462 (1990).)

These results suggest less protective methylation of 2-CE and morepronounced oxidation of CE to CE-Q in the kidney. To further investigatethe rationale behind this interpretation, hamsters were first pretreatedwith L-buthionine (SR)-sulfoximine, an inhibitor of GSH synthesis, todeplete GSH levels. The hamsters were then treated with E₂. Very lowlevels of CE and methoxyCE were observed in the kidney compared to theliver, suggesting little protective reduction of CE-Q to CE in thekidney (Table 3). Most significantly, the 4-OHE₁(E₂)-1-N7Guadepurinating adduct, arising from reaction of CE-3,4-Q with DNA, wasdetected in the kidney, but not in the liver (Table 3). From theseresults, it seems that tumor initiation in the kidney occurs because ofpoor methylation of CE, which favors the competitive oxidation of CE toCE-Q, and poor reductase activity to remove CE-Q. Thus, these twoeffects lead to a large amount of CE-Q, which can react with biologicalnucleophiles, including those in DNA. (Cavalieri E. L., et al., Chem.Res. Toxicol., 14, 1041-1050 (2001.) TABLE 3 Selected estrogenmetabolites, conjugates and adducts formed in hamsters treated with E₂or E₂ plus BSO^(a) nmol/g tissue Metabolites/conjugates^(b)/ KidneyLiver adducts E₂ E₂ + BSO E₂ E₂ + BSO 2-OHE₁(E₂) 2.66 1.02 4.75 10.274-OHE₁(E₂) 0.29 0.14 0.44 1.04 2-OCH₃E₁(E₂) 1.13 0.42 4.16 4.46E₁(E₂)-2,3-Q conjugates^(b) 1.36 0.21 0.63 0.13 E₁(E₂)-3,4-Qconjugates^(b) 0.30 0.09 0.06 0.01 E₁(E₂)-3,4-Q N7Gua <0.01 0.27 <0.01<0.01 adducts^(a)Data are from Cavalieri et al., Chem Res. Toxicol., 14: 10-41(2001). BSO: L-buthionine (SR)-sulfoximine. The notation E₁(E₂)indicates that the metabolites, conjugates or adducts of both E₁ and E₂are detected.^(b)Conjugates include all compounds produced by reaction of CE-Q withGSH and detected as GSH, cysteine or N-acetylcysteine conjugates.Studies in Estrogen Receptor-α Knockout (ERKO)/Wnt-I Mice.

A novel model for breast cancer was established by crossing micecarrying the Wnt-1 transgene (100% of adult females develop spontaneousmammary tumors) with the ERKO mouse line, in which the mice lackestrogen receptor-α and estrogen receptor-β is not detected in themammary tissue. Mammary tumors develop in these mice despite the lack offunctional estrogen receptor-α. To begin investigating whether estrogenmetabolite-mediated genotoxicity may play an important role in theinitiation of mammary tumors, the pattern of estrogen metabolites andconjugates was analyzed in ERKO/Wnt-1 mice. Extracts of hyperplasticmammary tissue and mammary tumors were analyzed by HPLC interfaced withan electrochemical detector. Picomole amounts of the 4-CE were detected,but their methoxy conjugates were not. Neither the 2-CE nor 2-methoxyCEwere detected. 4-CE-GSH conjugates or their hydrolytic products(conjugates of cysteine and N-acetylcysteine) were detected in picomoleamounts in both tumors and hyperplastic mammary tissue, demonstratingthe formation of CE-3,4-Q. These preliminary findings indicate thatestrogen homeostasis is unbalanced in the mammary tissue, in that thenormally minor 4-CE metabolites were detected in the mammary tissue, butnot the normally predominant 2-CE. In addition, methylation of CE wasnot detected, whereas formation of 4-CE-GSH conjugates was. (BocchinfusoW. P., et al., Cancer Res., 59, 1869-1876 (1999); Devanesan P, et al.,Carcinogenesis, 22, 1573-1576 (2001).)

Studies in Human Breast Tissue Specimens.

Imbalances in estrogen homeostasis were also observed in women withbreast carcinoma compared to women without breast cancer (Table 4).Breast tissue specimens obtained from women undergoing breast biopsy orsurgery were analyzed for 31 estrogen metabolites, conjugates anddepurinating DNA adducts by HPLC with electrochemical detection. Inwomen without breast cancer, a larger amount of 2-CE than 4-CE wasobserved. In women with breast carcinoma, the 4-CE were 3.5 times moreabundant than the 2-CE and were 4 times higher than in the women withoutbreast cancer. Furthermore, a statistically lower level of methylationwas observed for the CE in cancer cases compared to controls. Finally,the level of CE-Q conjugates in women with cancer was 3 times that incontrols, suggesting a larger probability for the CE-Q to react with DNAin the breast tissue of women with carcinoma. These data suggest thatinitiation of human breast cancer is due to imbalances in estrogenhomeostasis that result in excessive formation of the electrophilicCE-Q. In particular, the CE-3,4-Q can react with DNA to generatesuccessively depurinating adducts, apurinic sites and oncogenicmutations leading to breast cancer. (Badawi A. F., et al., Proc. Amer.Assoc. Cancer Res., 42, 664 (2001).) TABLE 4 Estrogen metabolites andconjugates in breast tissue from women with and without breast cancerCompounds,^(a) pmol/g tissue 4-OHE ¹ (E ² ) Breast Tissue 4-OHE₁(E₂)2-OHE₁(E)₂ 2-OHE₁(E₂) 4-OMeE₁(E₂) 2-OMeE₁(E₂) 4-+2-OMeE₁(E₂) CE-Qconjugates Controls^(b) 3.6 ± 2.1 6.9 ± 6.1 0.52 4.9 ± 1.8 3.6 ± 2.3 8.52.6 ± 1.3  (10)^(c) (25) (24) (16) (29) Breast cancer 14.7 ± 11.5 4.2 ±4.6 3.5 3.1 ± 2.3 1.7 ± 1.0 4.8 8.2 ± 6.4 cases^(b) (53) (46) (39) (29)(57) p^(d) 0.047 n.s.^(e) 0.049 0.050 0.003^(a)The notation E₁(E₂) indicates that the metabolites, conjugates oradducts of both E₁ and E₂ were detected.^(b)Controls include 18 women with benign breast tissue and 31 withbenign fibrocystic changes for a total of 49 women. Breast cancer casesinclude 28 women with carcinoma of the breast.^(c)Number in parentheses indicates the percentage of specimens in whichthe compound was detected.^(d)p was calculated by the student's t-test.^(e)n.s.: not significant.Unifying Mechanism of Initiation of Cancer and Other Diseases

Oxidation of catechols to semiquinones and quinones is a postulatedpathway to initiate cancer not only with endogenous estrogens but alsowith synthetic estrogens such as the human carcinogen diethylstilbestroland its hydrogenated derivative hexestrol. In fact, these two compoundsare also carcinogenic in the kidney of Syrian golden hamsters, and themajor metabolites are their catechols. These catechols may bemetabolically converted to catechol quinones. The catechol quinone ofhexestrol has chemical and biochemical properties similar to those ofCE-3,4-Q, namely, it specifically forms N7Gua and N3Ade adducts by1,4-Michael addition after reaction with dG or Ade, respectively, aswell as DNA (FIG. 8A). These data suggest that the hexestrol catecholquinone is the electrophile involved in tumor initiation by hexestrol.In turn, these results substantiate the hypothesis that CE-3,4-Q may bethe major endogenous tumor initiators. (Herbst A. L., et al., New Engl.J. Med., 284, 878-881 (1971); Li, J. J., et al., Cancer Res., 43,5200-5204 (1983); Liehr J. G., et al., Chem.-Biol. Interactions, 55,157-176 (1985); Haaf H. and Metzler M., Pharmacol., 34, 3107-3115(1985); Blaich, G., et al., J. Steroid Biochem., 35, 201-204 (1996);Metzler M. and McLachlan J. A., Adv. Exp. Med. Biol., 136A, 829-837(1981); Jan S.-T., et al., Chem. Res. Toxicol, 11, 412-419 (1998) andunpublished results).

The oxidation of phenols to catechols and then to semiquinones andquinones is not only a mechanism of tumor initiation for natural andsynthetic estrogens, but it could also be the mechanism of tumorinitiation for the leukemogen benzene (FIG. 8B). Certain metabolites ofbenzene may be responsible for both its cytotoxic and genotoxic effects.Benzene is metabolized to phenol in the liver by cytochrome P450 2E1.Other metabolites include catechol, hydroquinone (1,4-dihydroxybenzene)and muconaldehyde. Catechol and hydroquinone accumulate in the bonemarrow, where they can be oxidized by peroxidases, includingmyeloperoxidase and prostaglandin H synthase. The resulting quinones canyield DNA adducts. (Andrews, et al., Biochem. Pharmacol., 26, 293-300(1977); Sammett, D., et al., J. Toxicol. Environ. Health, 5, 785-792(1979); Snyder, R. and Kalf, G. F., CRC Crit. Rev. Toxicol., 24, 177-209(1994); Koop, D. R., et al., Toxicol. Appl. Pharmacol., 98, 278-288(1989); Guengerich, F. P., et al., Chem. Res. Toxicol., 4, 168-179(1991); Sabourin, P. J., et al., Toxicol. Appl. Pharmacol., 99, 421-444(1989); Latriano, L., et al., Proc. Natl. Acad. Sci. USA, 83, 8356-8360(1986); Schlosser, P. M., Carcinogenesis, 14, 2477-2486 (1993); Rickert,D. E., et al., Toxicol. Appl. Pharmacol., 49, 417-423 (1979); Greenlee,W. F., et al., Chem.-Biol. Interact., 33, 285-299 (1981); Eastmond, D.A., et al., Mol. Pharmacol., 30, 674-679 (1986); Subrahmanyam, V. V.,Arch. Biochem. Biophys., 286, 76-84 (1991); Sadler, A., et al., Toxicol.Appl. Pharmacol, 93, 62-71 (1988); Schlosser, M. J., et al., Chem. Res.Toxicol., 3, 333-339 (1990); Levay, G., et al., Carcinogenesis, 12,1181-1186 (1991); Levay, G. and Bodell, W. J., Proc. Natl. Acad. Sci.USA, 89, 7105-7109 (1992); Levay, G., et al., Carcinogenesis, 14,2329-2334 (1993).)

In fact, catechol, one of the metabolites of benzene, when oxidized tocatechol quinone, reacts with dG and Ade to form the catechol-4-N7Guaand catechol-4-N3Ade adducts in high yields, respectively. Oxidation ofcatechol catalyzed by horseradish peroxidase, tyrosinase orphenobarbital-induced rat liver microsomes in the presence of DNAyielded the catechol-4-N7Gua adduct, while the catechol-4-N3Ade adductwas obtained only with tyrosinase. (Balu N., et al., Proc. Amer. Assoc.Cancer Res., 40, 46 (1999); Cavalieri, E. L., et al., Carcinogenesis, inpress (2002).)

Catecholamine neurotransmitters such as dopamine may producesemiquinones and quinones via autoxidation, metal ion oxidation andperoxidative enzyme or cytochrome P450 oxidation. This oxidative processis similar to the one described for the benzene metabolite catechol andthe 4-CE, and it may initiate Parkinson's disease and otherneurodegenerative disorders. The etiology of Parkinson's disease and thebasic mechanism of loss of dopamine neurons are unknown. One of thefunctions of dopamine is the synthesis of neuronmelanin via oxidation ofdopamine to its quinone. If oxidation of dopamine to its quinone doesnot occur in a properly controlled environment, dopaamine quinone mayreact with DNA to cause damage by formation of specific depurinatingadducts. In fact, N7Gua and N3Ade adducts (FIG. 8B) are obtained byreaction of the dopamine quinone with dG or Ade, respectively, and thesame adducts are formed when dopamine is enzymatically activated in thepresence of DNA. The mutations generated by this damage may play a rolein the initiation of Parkinson's disease and other neurodegenerativedisorders. (Mattammal M. B., et al., J. Neurochem., 64, 1845-1854(1995); Kalyanaraman B., et al., Environ. Health Perspect., 64, 185-194(1985); Kalyanaraman B., et al., J. Biol. Chem., 259, 7584-7589 (1984);Balu N., et al., Proc. Amer. Assoc. Cancer Res., 40, 46 (1999);Cavalieri, E. L., Carcinogenesis, in press (2002)).

Conclusions

The carcinogenicity of estrogens in animal models led to aninvestigation of the plausible estrogen metabolites that could reactwith DNA and lead to mutations initiating cancer. The electrophilicCE-3,4-Q can, indeed, react with DNA to form the specific depurinatingadducts bonded at the N-7 of Gua and N-3 of Ade. The apurinic sitesformed by depurinating adducts are converted into tumor-initiatingmutations by error-prone repair. The specificity of the reaction of theelectrophiles with DNA is not limited to the natural estrogens, but alsoincludes the carcinogenic synthetic estrogens such as hexestrol. In thiscase metabolic formation of its catechol and further oxidation to itscatechol quinone lead to formation of analogous specific depurinatingadducts at the N-7 of Gua and N-3 of Ade. In addition, the metabolitecatechol of the leukomogenic benzene and the catecholamineneurotransmitter dopamine, when oxidized to quinone, binds to DNA toform N7Gua and N3Ade adducts. (Cavalieri E., et al., Estrogens asendogenous genotoxic agents: DNA adducts and mutations. In: JNCIMonograph 27: Estrogens as Endogenous Carcinogens in the Breast andProstate, 75-93, E. Cavalieri and E. Rogan (eds.), Oxford Press,Washington (2000); Chakravarti D., et al., Mutation Res., 456: 17-32(2000); Chakravarti D. et al., Oncogene, 20: 7945-7953 (2001); JanS.-T., et al., Chem. Res. Toxicol, 11: 412-419 (1998).)

Thus, a unifying mechanism, namely, formation of catechol quinones andreaction with DNA by 1,4-Michael addition to yield depurinating adducts,is at the origin of cancers induced by oxidation of endogenous andsynthetic estrogens, leukemia by oxidation of benzene, andneurodegenerative diseases by oxidation of dopamine. This unifyingmechanism provides targets for disease prevention and treatment, andmethods to assess risk of developing diseases and/or their progression.

EXAMPLE II

Catechol Ortho-Quinones: The Electrophilic Compounds That FormDepurinating DNA Adducts and Could Initiate Cancer and Other Diseases

Introduction

An important pathway in the metabolism of catechol estrogens (CE) andcatecholamines is the oxidation to their respective semiquinones andquinones. The basis of the biological activity of catechol quinones isrelated to their ability to act both as oxidants and electrophiles. Asoxidants, catechol quinones redox cycle with their semiquinones,producing an elevated level of reactive oxygen species, a conditionknown as oxidative stress. As electrophiles, catechol quinones can formcovalent adducts with cellular macromolecules, including DNA. These arestable adducts that remain in DNA unless removed by repair anddepurinating ones that are released from DNA by destabilization of theglycosyl bond. Thus, DNA can be damaged by the reactive quinonesthemselves and by reactive oxygen species (hydroxyl radicals). Theformation of depurinating adducts by CE quinones reacting with DNA maybe a major event in the initiation of breast and other human cancers.The depurinating adducts are released from DNA, leaving apurinic sitesin the DNA that can generate mutations leading to cancer. (Liehr, J. G.and Roy, D., Free Radic. Biol. Med., 8, 415-423 (1990); Cavalieri, E. L.and Rogan, E. G., The key role of catechol estrogen-3,4-quinones intumor initiation. In Creveling, C. R. (ed). Role of Catechol QuinoneSpecies in Cellular Toxicity, F.P. Graham Pub. Co., Johnson City, Tenn.,247-260 (2000); Finley, K. T., Quinones: The present state of additionand substitution chemistry. In Patai, S. (ed.) The chemistry ofhydroxyl, ether and peroxide groups, John Wiley & Sons Ltd., Suppl. E,1027-1134 (1993); Cavalieri, E. L., et al., Proc. Natl. Acad. Sci. USA,94, 10937-10942 (1997); Cavalieri, E., et al., Estrogens as endogenousgenotoxic agents: DNA adducts and mutations. In Cavalieri, E. and Rogan,E. (eds.) JNCI Monograph: “Estrogens as endogenous carcinogens in thebreast and prostate”, Oxford University Press, 75-93 (2000);Chakravarti, D., et al., Proc. Natl. Acad. Sci. USA, 92, 10422-10426(1995); Chakravarti, D., Mutat. Res., 456, 17-32 (2000); Chakravarti,D., et al., Oncogene, 20, 7945-7953 (2001).)

An important metabolic pathway of the estrogens, estrone (E₁) andestradiol (E₂), is formation of CE, namely, the hydroxylated estrogens,4-hydroxyestrone(estradiol) [4-OHE₁(E₂)], which are carcinogenic inanimals, and the isomeric 2-OHE₁(E₂). Oxidation of 4-OHE₁(E₂) to theirquinones [E₁(E₂)-3,4-Q] and reaction with DNA form the4-OHE₁(E₂)-1-N7guanine (Gua) and 4-OHE₁(E₂)-1-N3adenine (Ade) adducts bydepurination (FIG. 9). (Liehr, J. G., et al., J. Steroid Biochem., 24,353-356 (1986); Li, J. J. and Li, S. A., Fed. Proc., 46, 1858-1863(1987); Cavalieri, E. L., et al., Proc. Natl. Acad. Sci. USA, 94,10937-10942 (1997); Cavalieri, E., et al., Estrogens as endogenousgenotoxic agents: DNA adducts and mutations. In Cavalieri, E. and Rogan,E. (eds.) JNCI Monograph: “Estrogens as endogenous carcinogens in thebreast and prostate”, Oxford University Press, pp. 75-93 (2000); Li,K.-M., et al., Proc. Amer. Assoc. Cancer Res., 39, 636 (1998).)

Benzene

Benzene is carcinogenic and leukemogenic in rats and mice, andepidemiological studies have established a relationship between exposureto benzene and acute myelogenous leukemia in humans. Several studiesindicate that certain metabolites of benzene are responsible for bothits cytotoxic and genotoxic effects. High levels of peroxidase and alack of quinone reductase in the bone marrow allow formation of toxicsemiquinones and quinones without the possibility of their beingreduced. Benzene is initially metabolized to phenol in the liver bycytochrome P450 2E1. Other metabolites include catechol (CAT,1,2-dihydroxybenzene), hydroquinone (1,4-dihydroxybenzene) andmuconaldehyde. Several studies have shown that CAT and hydroquinoneaccumulate in bone marrow, where they can be further activated to exerttheir myelotoxic effects. (Cronkite, E. P., et al., Environ. HealthPerspect., 82, 97-108 (1989); Maltoni, C., et al., Environ. HealthPerspect., 82, 109-124 (1989); Huff, J. E., et al., Environ. HealthPerspect., 82, 125-163 (1989); (IARC, IARC Monographs on the Evaluationof the Carcinogenic Risk of Chemicals to Humans 29, 93-148 (1982);Rinsky, R. A., et al., N. Engl. J. Med., 316, 1044-1050 (1987); Andrews,L. S., et al., Biochem. Pharmacol., 26, 293-300 (1977); Sammett, D., etal., J. Toxicol. Environ. Health, 5, 785-792 (1979); Kalf, G. F., CRCCrit. Rev. Toxicol., 18, 141-159 (1987); Snyder, R. and Kalf, G. F., CRCCrit. Rev. Toxicol., 24, 177-209 (1994); Twerdok, L. E. and Trush, M.A., Res. Commun. Chem. Pathol. Pharmacol., 67, 375-386 (1990); Koop, D.R., et al., Toxicol. Appl. Pharmacol., 98, 278-288 (1989); Guengerich,F. P., et al, Chem. Res. Toxicol., 4, 168-179 (1991); Sabourin, P. J.,et al., Toxicol. Appl. Pharmacol., 99, 421-444 (1989); Latriano, L., etal., Proc. Natl. Acad. Sci. USA, 83, 8356-8360 (1986); Schlosser, P. M.,et al., Carcinogenesis, 14, 2477-2486 (1993); Rickert, D. E., et al.,Toxicol. Appl. Pharmacol., 49, 417-423 (1979); Greenlee, W. F., et al.,Chem.-Biol. Interact., 33, 285-299 (1981); Kalf, G. F., CRC Crit. Rev.Toxicol., 18, 141-159 (1987); Eastmond, D. A., Mol. Pharmacol., 30,674-679 (1986); Subrahmanyam, V. V., et al., Arch. Biochem. Biophys..286, 76-84 (1991); Sadler, A., et al., Toxicol. Appl. Pharmacol., 93,62-71 (1988); Schlosser, M. J., et al., Chem. Res. Toxicol., 3, 333-339(1990); Levay, G., et al., Carcinogenesis, 12, 1181-1186 (1991); Levay,G. and Bodell, W. J., Proc. Natl. Acad. Sci. USA, 89, 7105-7109 (1992);Levay, G., et al., Carcinogenesis, 14, 2329-2334 (1993).)

Dopamine

The neurotransmitter DA is formed in the cell bodies of the dopaminergicneurons of the substantia nigra. Degeneration of the nigrostriataldopaminergic neurons and decreased production of DA results inParkinson's disease. The etiology of Parkinson's disease and itsunderlying mechanism of loss of DA neurons are unknown. There isevidence, however, that DA is involved in the etiology of this disease,based on the observation by Graham, et al. that DA is oxidized to thecorresponding quinone. Injection of DA into neostriatum generatestoxicity to dopaminergic neurons, and the toxicity correlates withprotein binding. Glutathione and ascorbic acid diminish the toxicity ofprotein binding. Covalent binding of DA to DNA occurs upon incubating DAwith HL-60 cells or human glioblastoma cell lines, by copper-mediatedoxidation of DA or by oxidation of DA with prostaglandin H synthase.(Graham, D. G., et al., Mol. Pharmacol., 14, 644-653 (1978); Hastings,T. G., et al., Proc. Natl. Acad. Sci. USA, 93, 1956-1961 (1996);Filloux, F. and Townsend, J. J., Exper. Neurol., 119, 79-88 (1993);Hastings, T. G. and Zigmond, M. J., J. Neurochem., 63, 1126-1132 (1994);Levay, G. and Bodell, W. J., Carcinogenesis, 14, 1241-1245 (1993);Levay, G., et al., Exper. Neurol., 146, 570-574 (1997); Hastings, T. G.,J. Neurochem., 64, 919-924 (1995); Mattammal, M. B., J. Neurochem., 64,1845-1854 (1995).)

Oxidation of DA to its quinone and subsequent reaction with DNA maycause DNA damage via formation of specific depurinating adducts, and themutations generated by that damage may play a major role in initiatingthe series of events leading to neurodegenerative disorders such asParkinson's disease. In general, catecholamine neurotransmitters such asDA can produce semiquinones and quinones via autoxidation, metal ionoxidation, and peroxidative enzyme or cytochrome P450 oxidation. Thisoxidative process is similar to the one described above for the benzenemetabolite CAT and for the 4-OHE₁(E₂) formed by the metabolism of E₁ andE₂. (Kalyanaraman, B., et al., Environ. Health Perspect., 64, 185-194(1985); Kalyanaraman, B., et al., J. Biol. Chem., 259, 7584-7589 (1984);Cavalieri, E. L., et al., Proc. Natl. Acad. Sci. USA, 94, 10937-10942(1997); Cavalieri, E., et al., Estrogens as endogenous genotoxic agents:DNA adducts and mutations. In Cavalieri, E. and Rogan, E. (eds.) JNCIMonograph: “Estrogens as endogenous carcinogens in the breast andprostate”, Oxford University Press, 75-93 (2000).)

Abbreviations

Ade, adenine; o-BQ, ortho-benzoquinone; CAT, catechol or1,2-dihydroxybenzene; CE, catechol estrogen(s); CE-Q, catechol estrogenquinone(s); COMT, catechol-O-methyltransferase; DA, dopamine; dG,deoxyguanosine; DMF, dimethylformamide; E₁, estrone; E₂, estradiol;E₁(E₂)-3,4-Q, estrone(estradiol)-3,4-quinones or catecholestrogen-3,4-quinones; FAB, fast atom bombardment; Gua, guanine; MS/MS,tandem mass spectrometry; NADA, N-acetyldopamine; OHE₁(E₂),hydroxyestrone(estradiol); TFA, trifluoroacetic acid.

Definitions

The term catechol refers to an aromatic ring with vicinal hydroxylsubstituents. Herein catechol is spelled out when it is used in ageneral sense. When it refers specifically to the compound1,2-dihydroxybenzene, normally called catechol, it is abbreviated CAT.

Materials and Methods

Chemicals Reagents and Enzymes: CAT was obtained from ICNPharmaceuticals Inc., Cleveland, Ohio; Ag₂O, NaIO₄, Ade, thymidine,deuterated acetic acid and trifluoroacetic acid (TFA) were purchasedfrom Aldrich Chemical Co., Milwaukee, Wis. 2′-Deoxyguanosine (dG),2′-deoxyadenosine and 2′-deoxycytidine were purchased from TCIChemicals. DA, and N-acetyldopamine (NADA), horseradish peroxidase (typeVI) and mushroom tyrosinase were purchased from Sigma Chemicals, St.Louis, Mo. Liver microsomes from phenobarbital-induced female Wistar MRCrats (Eppley Colony) were prepared by the previously published method(Wong, A. K. L., et al., Biochem. Pharmacol., 35, 1583-1588 (1986).)

Instrumentation

UV: The UV spectra were obtained during HPLC by using the photodiodearray detector (Waters 996, Milford, Mass.) for all compoundssynthesized. HPLC separations were monitored at 280 nm.

NMR: Proton and homonuclear two-dimensional chemical shift correlationspectroscopy NMR spectra were recorded in DMSO-d₆ with one drop of D₂Oand one drop of CD₃COOD on a Varian Unity 500 instrument at 499.835 MHzat 25° C. Chemical shifts are reported relative to DMSO (2.5 ppm).

Mass spectrometry: Exact mass measurements of fast atom bombardment(FAB)-produced ions were carried out on a Kratos MS-50 double focusingmass spectrometer in a peak-match mode. Confirmation of the presence ofeach adduct was by capillary HPLC coupled via electrospray ionizationwith a Finnigan LCQ ion trap mass spectrometer operating in the tandemmass spectrometry (MS/MS) mode. The HPLC (Microtech Scientific) made useof a binary gradient of solvent A [0.5% CH₃COOH (v/v) in H₂O] andsolvent B [0.5% CH₃COOH (v/v) in CH₃OH] at a flow rate of 40 μL/min witha split of 10:1. The column was 0.3×100 Zorbax C18 (MicrotechScientific) with a flow rate on the column of 4 μL/min. The gradient was95% A/5% B initially for 4 min, then linearly adjusted to 60/40 over 14min, and held at 60/40 for 20 min.

HPLC methods for synthetic standards: HPLC was conducted on a Waters(Milford, Mass.) 600 E system equipped with a Waters 996 photodiodearray detector interfaced with an NEC-Powermate computer. Analyses andpreparative separations were carried out on reverse-phase C-18, YMC(Morris Plains, N.J.) columns (5 μm, 120A, ODS-AQ (6×250 mm) and ODS-AQ,5 μm, 120 Å, (20×250 mm), respectively) using specific mobile phases forthe different compounds.

Synthesis of Standard Adducts

Catechol adducts. Because the ortho-benzoquinone (o-BQ, nascent quinone)is rather unstable, various methods of synthesis were tested to obtainits maximum yield. Oxidation of CAT using Ag₂O in dry dimethylformamide(DMF) was the best method. A solution of CAT (100 mg, 0.91 mmol) in dryDMF (7.5 mL) was stirred with Ag₂O (842 mg, 3.60 mmol) for 30 min at 0°C. The extent of formation of o-BQ was followed by HPLC, using a linearanalytical gradient from 100% H₂O (0.01% TFA, pH 2.6) to 30% CH₃CN in 60min at a flow rate of 1 mL/min (monitored by UV absorbance at 300 nm ona Waters 996 photodiode array detector). The yield of o-BQ was >95%.

The dark red solution was immediately filtered into a solution of dG(1.20 g, 4.54 mmol) or Ade (613 mg, 4.54 mmol) in DMF/CH₃COOH/H₂O, 7.5mL each (FIG. 10). The reaction mixture was stirred for 8 h at roomtemperature, filtered and washed with 10 mL of DMF/CH₃COOH/H₂O (2:1:1).The reddish-brown filtrate was directly subjected to HPLC purification,using a linear preparative gradient of 20% CH₃CN in H₂O (0.01% TFA) to80% CH₃CN in H₂O (0.01% TFA) over 60 min at a flow rate of 9 mL/min withdG or 15% CH₃CN in H₂O (0.01% TFA) to 60% CH₃CN in H₂O (0.01% TFA) over60 min at a flow rate of 9 mL/min with Ade. The products, isolated underan argon atmosphere and stored at −20° C in 2 mL of DMF/CH₃COOH/H₂O(2:1:1), were CAT-4-N7Gua and CAT-4-N3Ade, the result of a 1,4-Michaeladdition between dG or Ade and o-BQ.

For CAT-4-N7Gua, the yield was 59%; UV: λ_(max) 285 nm. ¹H NMR, δ (ppm):6.79 (s, 2H, 5-H, 6-H), 7.08 (s, 1H, 3-H), 8.01 (s, 1H, 8-H [Gua]). FABMS, [M+H]⁺, Cl₁H₉N₅O₃: calcd m/z 260.0785; obsd m/z 260.0783.

For CAT-4-N3Ade, the yield was 65%; UV: λ_(max) 279 nm. ¹H NMR, δ (ppm):6.95 (bd, 2H, 5-H, 6-H), 7.08 (s, 1H, 3-H), 8.52 (s, 1H, 2-H [Ade]),8.67 (s, 1H, 8-H [Ade]). FAB MS, [M+H]⁺, C₁₁H₉N₅O₂: calcd m/z 244.0836;obsd m/z 244.0834.

N-Acetyldopamine adducts. A solution of NADA (9 mg, 0.047 mmol) in 1.5mL of CH₃COOH/H₂O (1:1) was stirred with NaIO₄ (5 mg, 0.023 mmol) for 5min at room temperature. To the resulting red solution of the NADAquinone was added 5 equivalents of dG (59 mg, 0.23 mmol) in 1.5 mL ofCH₃COOH/H₂O (1: 1) (FIG. 11). The reaction mixture was stirred for 3 hat room temperature and then separated by HPLC, using a 45-min linearpreparative gradient from 10% CH₃CN in H₂O (0.01% TFA) to 30% CH₃CN inH₂O (0.01% TFA) at a flow rate of 10 mL/min. The yield of NADA-6-N7Guawas 58%.

The NADA-6-N7Gua adduct was also synthesized following oxidation of NADAby Ag₂O. A solution of NADA (5 mg, 0.023 mmol) in 1 mL of dry DMF wasstirred with Ag₂O (43 mg, 0.19 mmol) for 30 min. The suspension wasimmediately filtered into a solution of dG (29 mg, 0.12 mmol) inDMF/CH₃COOH/H₂O, 1 mL each. The reaction mixture was stirred for 10 h atroom temperature, and the product purified by HPLC, yielding 60%NADA-6-N7Gua, UV: λ_(max), 245, 284 nm. ¹H NMR, δ (ppm): 1.90 (s, 3H,CH₃), 2.25 (t, 2H, J=6.7 Hz, 7-CH₂), 3.00 (bt, 2H, 8-CH₂), 6.62 (s, 1H,5-H), 6.66 (s, 1H, 2-H), 7.85 (s, 1H, 8-H [Gua]). FAB MS, [M+H]⁺,C₁₅H₁₇N₆O₄: calcd m/z 345.1311; obsd m/z 345.1311.

To synthesize the Ade adduct, a solution of NADA (20 mg, 0.094 mmol) in2 mL of CH₃COOH/H₂O (1:1) was oxidized with NaIO₄ (10 mg, 0.047 mmol)and reacted with Ade (63 mg, 0.47 mmol), as described above for thereaction with dG. The product, NADA-6-N3Ade, was purified by HPLC, usinga preparative linear gradient from 5% CH₃CN in H₂O (0.01% TFA) over 60min to 40% CH₃CN in H₂O (0.01% TFA) at a flow rate of 9 mL/min. Theyield was 51%; UV: λ_(max), 275 nm. ¹H NMR, δ (ppm): 1.90 (s, 3H, CH₃),2.15-2.30 (m, 2H, 7-CH₂), 2.83-3.10 (m, 2H, 8-CH₂), 6.81 (s, 1H, 5-H),6.85 (s, 1H, 2-H), 8.45 (s, 1H, 2-H [Ade]), 8.65 (s, 1H, 8-H [Ade]). FABMS, [M+H]⁺, C₁₅H₁₇N₆O₃: calcd m/z 329.1361; obsd m/z 329.1362.

Dopamine adducts. DA·HCl (50 mg, 0.264 mmol) and dG (622 mg, 2.6 mmol)were dissolved in 13 mL of CH₃COOH/H₂O (1:1). To this mixture a solutionof NaIO₄ (28 mg, 0.13 mmol) in 2 mL of CH₃COOH/H₂O (1:1) was addeddropwise over 10 min (FIG. 11). After 3 h at room temperature, thereaction was terminated, and the product, DA-6-N7Gua, was purified bypreparative HPLC, using a linear gradient from 10% CH₃CN in H₂O (0.01%TFA) to 30% CH₃CN in H₂O (0.01% TFA) over 60 min, then to 80% CH₃CN in15 min at a flow rate of 8 mL/min. The colorless semi-solid product wasobtained in 46% yield. UV: λ_(max), 245 (sh), 283 nm. ¹H NMR, δ (ppm):2.45 (m, 2H, 7-CH₂), 2.75 (m, 2H, 8-CH₂), 6.70 (s, 1H, 5-H), 6.70 (s,1H, 2-H), 8.15 (s, 1H, 8-H [Gua]). FAB MS, [M+H]⁺, C₁₃H₁₄N₆O₃: calcd m/z303.1207; obsd m/z 303.1205.

A solution of DA·HCl (50 mg, 0.26 mmol) and Ade (356 mg, 2.64 mmol) in13 mL of CH₃COOH/H₂O (1:1) was treated with a solution of NaIO₄ (28 mg,0.13 mmol) in 2 mL of CH₃COOH/H₂O (1:1) in a manner similar to that usedto synthesize DA-6-N7Gua. After 3 h at room temperature, the reactionwas terminated. The mixture was subjected to preparative HPLC, using H₂O(0.01% TFA) at a flow rate of 5 mL/min for 20 min, followed by a lineargradient to 80% CH₃CN in H₂O (0.01% TFA) over 40 min at a flow rate of10 mL/min. DA-6-N3Ade was obtained in 40% yield. UV: λ_(max) 279 nm. ¹HNMR, δ (ppm): 2.40 (m, 2H, 7-CH₂),2.82 (m, 2H, 8-CH₂), 6.80 (s, 1H,5-H), 6.86 (s, 1H, 2-H), 8.05 (s, 1H, 2-H [Ade]), 8.40 (s, 1H, 8-H[Ade]). FAB MS, [M+H]⁺, C₁₃H₁₄N₆O₂: calcd m/z 287.1258; obsd m/z287.1256.

Enzymatically-Catalyzed Covalent Binding of Catechol and Dopamine to DNA

CAT and DA were bound to DNA in 10-mL reaction mixtures containing 3 mMcalf thymus DNA in 0.067 M sodium-potassium phosphate (pH 7.0), 0.8 μMCAT or DA in 50 μL DMSO and 1 mg horseradish peroxidase plus 0.5 mM H₂O₂or 1 mg mushroom tyrosinase. CAT and DA (0.8 μM) were also activated by10 mg of phenobarbital-induced rat liver microsomes in 150 mM Tris-HCl(pH 7.5), 150 mM KCl, 5 mM MgCl₂, 1 mM cumene hydroperoxide and 3 mMDNA. The reactions were incubated for 2 h at 37° C. A 1-mL aliquot wasused for analysis of stable DNA adducts by the ³²P-postlabeling methodwith 8 μg of DNA. The DNA was precipitated from the remaining reactionmixture with two volumes of ethanol, and the supernatant was used forstructure determination of depurinating adducts. After evaporation undervacuum, the residue was dissolved in 1 mL of DMSO/CH₃OH. The CAT adductswere first separated by HPLC on a preparative column with a curvilineargradient (CV 6) from 100% H₂O (0.01% TFA) to 15% CH₃OH in H₂O (0.01%TFA) in 60 min at a flow rate of 3 mL/min. Fractions at the retentiontimes of CAT-4-N3Ade (34.5 min) and CAT-4-N7Gua (40 min) were collectedand analyzed by HPLC, which was eluted with aqueous 50 mM (NH₄)₃PO₄, 5mM sodium dodecyl sulfate, 1% CH₃COOH at a flow rate of 0.5 mL/min. TheDA adducts were first separated by HPLC on a preparative column elutedwith a curvilinear gradient (CV 6) from 100% aqueous 50 mM (NH₄)₃PO₄, 5mM sodium dodecyl sulfate, 4% CH₃COOH to 100% CH₃CN at a flow rate of0.5 mL/min. Fractions were collected at 34 min for DA-6-N3Ade and 37 minfor DA-6-N7Gua and analyzed by HPLC as described above for the CATadducts. The remainder of the collected fractions was used to confirmthe structures of the adducts by MS. (Wong, A. K. L., et al., Biochem.Pharmacol., 35, 1583-1588 (1986); Bodell, W. J., et al., Chem. Res.Toxicol., 2, 312-315 (1989).)

Results

To demonstrate that the quinones of CAT and DA can react with thenucleobases of DNA, standard adducts were synthesized by reaction ofo-BQ or DA quinone with dG or Ade. The syntheses provided usefulinsights into the ability of these electrophilic species to react withnucleophilic groups of deoxyribonucleosides. Furthermore, the adductsserved as standards to identify the depurinating adducts formed when CATand DA were oxidized in vitro by various enzymes in the presence of DNA(see below).

Structure Elucidation of Adducts

Catechol adducts. The reaction of o-BQ with the deoxyribonucleosidebases to afford the desired adducts is an acid-assisted 1,4-Michaeladdition reaction analogous to that of CE quinones with nucleobases.With o-BQ, however, the reaction in CH₃COOH/H₂O (1:1) did not yield anyproducts, due to the instability of o-BQ. To render this reactionfeasible a compromise was reached by conducting it in DMF/CH₃COOH/H₂O(2:1:1). (Stack, D.,et al., Chem. Res. Toxicol., 9, 851-859 (1996); Jan,S.-T., et al., Chem. Res. Toxicol., 11, 412-419 (1998).)

Reaction of o-BQ with dG afforded CAT-4-N7Gua (FIG. 10). The structurewas readily determined by both NMR and MS analysis. By MS, the [M+H]⁺ion had an m/z 260, indicating that deoxyribose had been lost. Thisimplies that Gua is bonded to CAT at the N-7. The NMR resonance of the5-H and 6-H of the CAT moiety as a singlet at 6.79 ppm and the 3-H as asinglet at 7.08 ppm indicates that the bond to Gua in the CAT aromaticring occurs at C-4.

For CAT-4-N3Ade, the structure was consistent with the NMR spectrum,showing the aromatic protons 5-H and 6-H as a doublet at 6.95 ppm, andthe singlet at 7.08 ppm that was assigned as 3-H. Furthermore, the 2-Hand 8-H of the Ade moiety were observed at 8.52 and 8.76 ppm,respectively. The mass of the FAB-produced ion at m/z 244 corroboratedthe structure of this adduct.

Under the same conditions, o-BQ adducts of deoxyadenosine, deoxycytidineand thymidine were not obtained. Furthermore, reaction of the stable1,4-benzoquinone (p-BQ) with dG, Ade, deoxyadenosine, deoxycytidine orthymidine did not afford any detectable adducts.

Dopamine adducts. The oxidation of DA and subsequent reaction with dG orAde were more difficult to accomplish because the amino group of the DAquinone reacts intramolecularly by a 1,4-Michael addition to produce adihydroindole, a precursor to neuromelanin biosynthesis. This reactioncompetes with the intermolecular acid-assisted 1,4-Michael addition ofthe nucleophilic groups of dG and Ade to the DA quinone. To avoid thecompetitive cyclization reaction, NADA, in which the nucleophilic aminogroup of DA is acetylated, was oxidized to its quinone and reacted withdeoxyribonucleosides or nucleobases.

N-Acetyldopamine. The structure of the NADA-6-N7Gua adduct (FIG. 11) wasconsistent with MS results, which showed an m/z 345 ion, indicating theloss of the deoxyribose moiety. It was also consistent with the NMRresults: the two aromatic protons of the NADA moiety, 5-H and 2-H,resonate at 6.62 and 6.70 ppm, respectively, assuming that the reactionwas a 1,4-Michael addition. If the reaction occurred by 1,6-addition,however, an adduct at C-2 and/or C-5 of NADA should have been obtained.Reaction at C-2 can be disregarded on the basis that the aromaticprotons did not resonate as doublets. The adduct with the Gua-NADA bondat C-5, formed by 1,6-addition, was eliminated from consideration by anuclear Overhauser enhancement experiment in which the resonance of the7-CH₂ protons at 2.25 ppm was irradiated. This structure would entailthat both the resonance signals corresponding to 2-H and 6-H beenhanced. In fact, only the signals corresponding to the 2-H of NADA at6.70 ppm and the 8-H of the Gua moiety at 7.85 ppm were enhanced. Thisresult unequivocally assigns the structure of the adduct asNADA-6-N7Gua, proving that the reaction is a 1,4-Michael addition.Following the same approach, the structure of NADA-6-N3Ade was assigned.

Dopamine. Reactions of DA-HCl were set at pH 1.2 in CH₃COOH/H₂O (1:1) tominimize intramolecular 1,4-Michael addition of DA and favorintermolecular 1,4-Michael addition of dG or Ade to the 6 position of DAquinone. Although cyclization was avoided under these conditions, owingto the extensive protonation of the DA amino group, minor side reactionssuch as dimerization and subsequent oligomerization of the resulting DAquinone could not be eliminated. To minimize these competing reactionsand obtain the best yields, the DA o-quinone was generated in situ byadding a solution of NaIO₄ to a mixture of DA-HCl and dG or Ade.

The structures of the adducts obtained by reaction of DA quinone with dGor Ade, DA-6-N7Gua and DA-6-N3Ade (FIG. 11), were elucidated followingthe same criteria adopted for the NADA adducts. Under the aboveconditions, DA quinone did not react with deoxyadenosine, deoxycytidineor thymidine to form adducts to any measurable extent.

In conclusion, the reaction of CAT quinone and DA quinone with dG or Adeinvolves the specific nucleophilic sites of the N-7 of Gua and N-3 ofAde in the 1,4-Michael addition. The reactions of E₁(E₂)-3,4-Q with dGor Ade by 1,4-Michael addition exhibit the same specificity, formingN7Gua and N3Ade depurinating adducts (Li, K.-M., et al., Proc. Amer.Assoc. Cancer Res., 39, 636 (1998); Stack, D., Chem. Res. Toxicol., 9,851-859 (1996).)

Enzymatically-Catalyzed Covalent Binding of Catechol and Dopamine to DNA

Conversion of CAT and DA to their quinones can generally occur byautoxidation, metal-ion oxidation or cytochrome P450 orperoxidase-catalyzed oxidation. In vivo the copper-containing enzymetyrosinase oxidizes DA to its quinone. To demonstrate binding to DNA invitro, CAT and DA were oxidized in reactions catalyzed by horseradishperoxidase, tyrosinase or phenobarbital-induced rat liver microsomes inthe presence of DNA (Table 5). TABLE 5 Catechol- and Dopamine-DNAAdducts Formed In Vitro. μmol adduct/mol DNA-P^(a) Phenobarbital-Horseradish induced rat Adduct Peroxidase liver microsomes TyrosinaseCatechol CAT-4-N7Gua 10 32 110 CAT-4-N3Ade nd^(b) nd 2 Stable adducts0.64 0.02 0.21 Dopamine DA-6-N7Gua 1 23 6 DA-6-N3Ade 5 9 3 Stableadducts 0.30 0.24 0.35^(a)Values are the average of two determinations that varied by 10-20%.^(b)nd: not detectedAll three enzymes catalyzed formation of detectable amounts of thedepurinating adducts of DA, DA-6-N3Ade and DA-6-N7Gua, as well as theCAT-4-N7Gua depurinating adduct of CAT. In contrast, the CAT-4-N3Adeadduct was detected only after activation by tyrosinase. (Kalyanaraman,B., et al., Environ. Health Perspect., 64, 185-194 (1985); Kalyanaraman,B., et al., J. Biol. Chem., 259, 7584-7589 (1984).)

Formation of the stable adducts of DA was low, less than 5% of the totaladducts formed with horseradish peroxidase, 4% of the adducts formedwith tyrosinase, and 1% of the adducts formed with microsomes.Similarly, with CAT, stable adducts comprised less than 6% of the totaladducts formed with horseradish peroxidase, 0.2% of those formed withtyrosinase, and 0.1% of the adducts formed with microsomes. With DA, themicrosomes catalyzed formation of six to seven stable adducts that wereseparated by the ³²P-postlabeling method, whereas tyrosinase andhorseradish peroxidase catalyzed formation of the same stable adduct,which appeared to be one of those formed by the microsomes. With CAT,both the microsomes and horseradish peroxidase formed two adductsseparated by ³²P-postlabeling. One of these adducts was detected withactivation by both enzymes. This same adduct was the only stable adductdetected when tyrosinase was used to catalyze the binding of CAT to DNA.

Confirmation of the presence of each depurinating adduct reported inTable 5 was by capillary HPLC/tandem mass spectrometry. The unknownshave identical HPLC retention times as the standards and giveproduct-ion spectra of the [M+H]⁺ ions containing the same two or threeintense signals as those of the standards. The product-ion spectrum ofthe two modified guanine [M+H]⁺ ions showed that losses of 17 (NH₃) and42 (NC-NH₂) occurred for both. The CAT-modified Gua showed an additionalloss of 24 (possible via formation of an ion-molecule product in thetrap), whereas the DA-modified Gua underwent a loss of 35 (NH₃ and H₂O).The modified bases isolated from the in vitro experiments showed thesesame ions. The [M+H]⁺ ions of the adenines modified with CAT or DAfragmented by losses of 17 and 46, and the unknowns also showed signalsfor these processes. The product-ion spectra of all the in vitro adductsshowed other comparable or weaker signals owing to coelutinginterferences from the reaction mixture.

In summary, enzymatic oxidation of CAT or DA in the presence of DNAresulted in the formation of 94-99.9% depurinating CAT adducts or 95-99%depurinating DA adducts.

Discussion

The catechol o-quinones derived from benzene and DA undergo 1,4-Michaeladdition with the N-7 and N-3 nucleophilic sites of Gua and Ade in DNA,respectively, to form predominantly depurinating adducts analogous tothose formed by the E₁(E₂)-3,4-Q (FIG. 9). These depurinating adductsare by far the major products (94-99.9%) when the two o-quinones areenzymatically obtained from the corresponding catechols, CAT and DA, inthe presence of DNA (Table 5). (Cavalieri, E. L., et al., Proc. Natl.Acad. Sci. USA, 94, 10937-10942 (1997); Li, K.-M., et al., Proc. Amer.Assoc. Cancer Res., 39, 636 (1998).)

The role of estrogens in causing DNA damage is better understood thanthat of CAT and DA. The estrogens E₁ and E₂, which are biochemicallyinterconvertible, are metabolized via two major pathways: formation ofCE and, to a lesser extent, 16α-hydroxylation. In general, estrogens andCE are inactivated by conjugating reactions, such as glucuronidation andsulfation, especially in the liver. The most common pathway of CEconjugation in extrahepatic tissues is O-methylation catalyzed by theubiquitous catechol-O-methyltransferase (COMT, FIG. 9). Relatively highlevels of cytochrome P450 1B1 and other 4-hydroxylases could cause the4-OHE₁(E₂), which are usually minor metabolites, to be the major ones,rendering conjugation of 4-OHE₁(E₂) via methylation in extrahepatictissues insufficient. In this case, competitive catalytic oxidation ofCE to CE quinones could occur (FIG. 9). Redox cycling generated byreduction of CE-Q to CE semiquinones, catalyzed by cytochrome P450reductase, and subsequent oxidation back to CE-Q by molecular oxygencauses formation of superoxide anion radicals and, subsequently,hydroxyl radicals (not shown in FIG. 9). This process, which also occurswith the quinones of CAT and DA, may constitute a significant source ofreactive oxygen species. Hydroxyl radicals can also react with DNA andcontribute to total DNA damage. (Cavalieri, E., et al., Estrogens asendogenous genotoxic agents: DNA adducts and mutations. In Cavalieri, E.and Rogan, E. (eds.) JNCI Monograph: “Estrogens as endogenouscarcinogens in the breast and prostate”, Oxford University Press, 75-93(2000); Cavalieri, E. L., et al., Proc. Natl. Acad. Sci. USA, 94,10937-10942 (1997); Service, R., Science, 279, 1631-1633 (1998); Liehr,J. G. and Roy, D., Free Radic. Biol. Med., 8, 415-423 (1990).

CE-Q can be inactivated by conjugation with glutathione (FIG. 9). Asecond inactivating pathway for CE-Q is their reduction to CE by quinonereductase and/or cytochrome P450 reductase (FIG. 9). If the twoinactivating processes are insufficient, CE-Q may react with DNA to formpredominantly stable adducts for the 2-OHE₁(E₂) (not shown in FIG. 9)and predominantly depurinating adducts for the 4-OHE₁(E₂) (FIG. 9). Thedepurinating adducts generate apurinic sites that may lead to oncogenicmutations, thereby initiating a variety of human cancers, includingbreast and prostate. In support of this hypothesis, a burst of apurinicsites leads to mutations in the H-ras gene of mouse skin treated withE₂-3,4-Q. (DT Diaphorase—A quinone reductase with special functions incell metabolism and detoxification. Ernester, L., Estabrook, R. W., etal.. (eds.) Chemica Scripta. 27A (1987); Roy, D. and Liehr, J. G., J.Biol. Chem., 263, 3646-3651 (1988); Cavalieri, E. L., et al., Proc.Natl. Acad. Sci. USA, 94, 10937-10942 (1997); Stack, D., et al., Chem.Res. Toxicol., 9, 851-859 (1996); Dwivedy, I., et al., Chem. Res.Toxicol., 5, 828-833 (1992); Li, K.-M., et al., Proc. Amer. Assoc.Cancer Res., 39, 636 (1998); Chakravarti, D., et al., Proc. Natl. Acad.Sci. USA, 92, 10422-10426 (1995); Chakravarti, D., et al., Mutat. Res.,456, 17-32 (2000); Chakravarti, D., Oncogene, 20, 7945-7953 (2001).)

The initiating mechanism of carcinogenesis for the synthetic estrogenhexestrol may have a similar explanation. This compound, which iscarcinogenic in the kidney of Syrian golden hamsters, also has catecholas a major metabolite, which can be metabolically converted to catecholquinone. The catechol quinone of hexestrol has chemical propertiessimilar to those of E₁(E₂)-3,4-Q, namely, it specifically forms an N7Guaadduct by 1,4-Michael addition after reaction with dG or DNA. (Li, J.J., Cancer Res., 43, 5200-5204 (1983); Liehr, J. G., et al., Chem.-Biol.Interactions, 55, 157-176 (1985); Metzler, M. and McLachlan, J. A., Adv.Exp. Med. Biol., 136A, 829-837 (1981); Jan, S.-T., Chem. Res. Toxicol.,11, 412-419 (1998).

The formation of depurinating adducts specifically at the N-7 of Gua andN-3 of Ade by 1,4-Michael addition to CAT quinone, analogously to thoseformed by E₁(E₂)-3,4-Q, suggests that the metabolite CAT may play amajor role in tumor initiation by benzene. In fact, CAT is carcinogenicin mice and rats, inducing glandular stomach tumors in these animals.The overall leukemogenicity of benzene could result from a synergisticgenotoxic response to CAT quinone, which predominantly producesdepurinating DNA adducts, and 1,4-benzoquinone, which produces onlystable DNA adducts. (Hirose, M., et al., Carcinogenesis, 14, 525-529(1993); Levay, G., et al., Carcinogenesis, 12, 1181-1186 (1991); Levay,G. and Bodell, W. J., Proc. Natl. Acad. Sci. USA, 89, 7105-7109 (1992);Robertson, M., et al., Mutat. Res., 249, 201-209 (1990); Smith, M. T.,Environ. Health Perspect., 104: Suppl. 6, 1219-1225 (1996).)

One of the functions of the neurotransmitter DA or its precursor,L-Dopa, is the synthesis of neuromelanin. This occurs by oxidation of DAto its o-quinone, followed by intramolecular cyclization of thenucleophilic amino group via a 1,4-Michael addition (FIG. 12). Theproduct, leucochrome, is further oxidized to aminochrome, which, aftertautomerization to its quinone methide and quinone imine, polymerizes toneuromelanin, the pigment of the substantia nigra. Disregulation of DAcompartmentalization may lead to DA quinone formation by variousoxidants. Under these circumstances, intermolecular 1,4-Michael additionof the N-7 of Gua or N-3 of Ade in DNA to DA quinone could competesuccessfully with the intramolecular cyclization of DA quinone thatleads to dihydroindole derivatives (FIG. 12). In fact, DA cyclizes at aslower rate than L-Dopa and epinephrine. Thus, if oxidation of DA to itsquinone does not occur in a properly controlled environment, thenperhaps the quinone will react with DNA to form depurinating DNAadducts, generating mutations that could initiate neurodegenerativedisorders such as Parkinson's disease. (Hastings, T. G., J. Neurochem.,64, 919-924 (1995); Mattammal, M. B., et al., J. Neurochem., 64,1845-1854 (1995); Kalyanaraman, B., et al, Environ. Health Perspect.,64, 185-194 (1985); Kalyanaraman, B., et al., J. Biol. Chem., 259,7584-7589 (1984); Pelizzetti, E., et al., J. Chem. Soc. Perkins II,1651-1655 (1976).)

Conclusions

The o-benzoquinones formed in the metabolism of natural and syntheticestrogens, benzene, and DA react with DNA via 1,4-Michael addition toform specific depurinating adducts that may lead to critical mutationsresponsible for initiating many cancers and neurodegenerative diseases.Recognition of this proposed unifying mechanism in the etiology of thesediseases may provide unique opportunities to develop strategies toassess risk and to prevent diseases.

All publications, patents, and patent documents are incorporated byreference herein, as though individually incorporated by reference. Theinvention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

1. (canceled)
 2. A method for preventing or reducing the risk ofneurodegenerative disease in a mammal comprising administering to themammal a therapeutically effective amount of the pharmaceuticalcomposition of claim
 33. 3. A method for preventing or reducing the riskof cancer in a mammal comprising administering to the mammal atherapeutically effective amount of the pharmaceutical composition ofclaim
 33. 4. A method for preventing or reducing the risk ofneurodegenerative disease and cancer in a mammal comprisingadministering to the mammal the pharmaceutical composition of claim 33.5. (canceled)
 6. (canceled)
 7. (canceled)
 8. (canceled)
 9. (canceled)10. (canceled)
 11. (canceled)
 12. (canceled)
 13. (canceled) 14.(canceled)
 15. (canceled)
 16. (canceled)
 17. (canceled)
 18. (canceled)19. (canceled)
 20. (canceled)
 21. (canceled)
 22. (canceled) 23.(canceled)
 24. (canceled)
 25. (canceled)
 26. (canceled)
 27. (canceled)28. (canceled)
 29. (canceled)
 30. (canceled)
 31. (canceled) 32.(canceled)
 33. A pharmaceutical composition comprising 1) NAC or aphysiologically acceptable salt thereof, 2) melatonin or aphysiologically acceptable salt thereof, 3) optionally one or moreagents that induce quinone reductase, and 4) one or more physiologicallyacceptable carriers or excipients.
 34. The composition of claim 33 thatcomprises lipoic acid or a pharmaceutically acceptable salt thereof. 35.The composition of claim 33 that comprises resveratrol or apharmaceutically acceptable salt thereof.
 36. The composition of claim34 that comprises resveratrol or a pharmaceutically acceptable saltthereof.
 37. A method for preventing or reducing the risk ofneurodegenerative disease in a mammal comprising administering to themammal a therapeutically effective amount of the pharmaceuticalcomposition of claim
 34. 38. A method for preventing or reducing therisk of neurodegenerative disease in a mammal comprising administeringto the mammal a therapeutically effective amount of the pharmaceuticalcomposition of claim
 35. 39. A method for preventing or reducing therisk of neurodegenerative disease in a mammal comprising administeringto the mammal a therapeutically effective amount of the pharmaceuticalcomposition of claim
 36. 40. A method for preventing or reducing therisk of cancer in a mammal comprising administering to the mammal atherapeutically effective amount of the pharmaceutical composition ofclaim
 34. 41. A method for preventing or reducing the risk of cancer ina mammal comprising administering to the mammal a therapeuticallyeffective amount of the pharmaceutical composition of claim
 35. 42. Amethod for preventing or reducing the risk of cancer in a mammalcomprising administering to the mammal a therapeutically effectiveamount of the pharmaceutical composition of claim
 36. 43. A method forpreventing or reducing the risk of neurodegenerative disease and cancerin a mammal comprising administering to the mammal the pharmaceuticalcomposition of claim
 34. 44. A method for preventing or reducing therisk of neurodegenerative disease and cancer in a mammal comprisingadministering to the mammal the pharmaceutical composition of claim 35.45. A method for preventing or reducing the risk of neurodegenerativedisease and cancer in a mammal comprising administering to the mammalthe pharmaceutical composition of claim
 36. 46. A method for preventingor reducing the risk of neurodegenerative disease by administering 1)NAC or a physiologically acceptable salt thereof, 2) melatonin, 3)lipoic acid, 4) resveratrol together or in any combination thereof. 47.A method for preventing or reducing the risk of cancer byadministering 1) NAC or a physiologically acceptable salt thereof, 2)melatonin, 3) lipoic acid, 4) resveratrol together or in any combinationthereof.
 48. A method for preventing or reducing the risk ofneurodegenerative disease and cancer by administering 1) NAC or aphysiologically acceptable salt thereof, 2) melatonin, 3) lipoic acid,4) resveratrol together or in any combination thereof.